Solid phase native chemical ligation of unprotected or n-terminal cysteine protected peptides in aqueous solution

ABSTRACT

The present invention provides methods, apparatus and kits for synthesizing assembled peptides and proteins on a solid phase with sequential ligation of three or more unprotected peptide segments using chemoselective and mild ligation chemistries in aqueous solution. Also provided are methods of monitoring solid phase sequential ligation reactions using MALDI or electrospray ionization mass spectrometry of reaction products.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/049,553, filed Jun. 13, 1997.

INTRODUCTION Background

Existing methods for the chemical synthesis of proteins include stepwisesolid phase synthesis, and fragment condensation either in solution oron solid phase. The classic stepwise solid phase synthesis of Merrifieldinvolves covalently linking an amino acid corresponding to thecarboxy-terminal amino acid of the desired peptide chain to a solidsupport and extending the polypeptide chain toward the amino end bystepwise coupling of activated amino acid derivatives having activatedcarboxyl groups. After completion of the assembly of the fully protectedsolid phase bound peptide chain, the peptide-solid phase covalentattachment is cleaved by suitable chemistry and the protecting groupsremoved to give the product polypeptide.

Some disadvantages of the stepwise solid phase synthesis method include:incomplete reaction at the coupling and deprotection steps in each cycleresults in formation of solid-phase bound by products. Similarly, sidereactions due to imperfections in the chemistry, and or impuritiespresent in the reagents/protected amino acids, all lead to amultiplicity of solid phase bound products at each step of the chainassembly and to the formation of complex product mixtures in the finalproduct. Thus, the longer the peptide chain, the more challenging it isto obtain high-purity well-defined products. Due to the production ofcomplex mixtures, the stepwise soid phase synthesis approach has sizelimitations. In general, well-defined polypeptides of 100 amino acidresidues or more are not routinely prepared via stepwise solid phasesynthesis. Synthesis of proteins and large polypeptides by this route isa time-consuming and laborious task.

The solid phase fragment condensation approach (also known as segmentcondensation) was designed to overcome the difficulties in obtaininglong polypeptides via the solid phase stepwise synthesis method. Thesegment condensation method involves preparation of several peptidesegments by the solid phase stepwise method, followed by cleavage fromthe solid phase and purification of these maximally protected segments.The protected segments are condensed one-by-one to the first segment,which is bound to the solid phase.

Often, technical difficulties are encountered in many of the steps ofsolid phase segment condensation. See E. Atherton, et al., “Solid PhaseFragment Condensation—The Problems,” in Innovation and Perspectives inSolid Phase Synthesis 11-25 (R. Epton, et al. 1990). For example, theuse of protecting groups on segments to block undesired ligatingreactions can frequently render the protected segments sparinglysoluble, interfering in efficient activation of the carboxyl group.Limited solubility of protected segments also can interfere withpurification of protected segments. See K. Akaji et al., Chem. Pharm.Bull.(Tokyo) 33:184-102 (1985). Protected segments are difficult tocharacterize with respect to purity, covalent structure, and are notamenable to high resolution analytical ESMS (electrospray massspectrometry) (based on charge). Racemization of the C-terminal residueof each activated peptide segment is also a problem, except if ligatingis performed at Glycine residues. Moreover, cleavage of the fullyassembled, solid-phase bound polypeptide from the solid phase andremoval of the protecting groups frequently can require harsh chemicalprocedures and long reaction times that result in degradation of thefilly assembled polypeptide.

Segment condensation can be done in solution rather than on solid phase.See H. Muramatsu et al., Biochem. and Biophys. Res. Commn.203(2):1131-1139 (1994). However, segment condensation in solutionrequires purification of segments prior to ligation as well as use ofprotecting groups on a range of different side chain functional groupsto prevent multiple undesired side reactions. Moreover, the ligation insolution does not permit easy purification and wash steps afforded bysolid phase ligations. Furthermore, the limitations with respect tosolubility of protected peptide segments and protected peptideintermediate reaction products are exacerbated.

Chemical ligating of minimally protected peptide segments has beenexplored in order to overcome the solubility problems frequentlyencountered with maximally protected peptide segments. See Cheng, etal., Chemical Synthesis of Human -endorphin(1-27) Analogs by PeptideSegment Coupling. Int. J. Pept. Protein Res. 38:70-78 (1991); J. Blake,Total Synthesis of S-Carbamoylmethyl Bovine Apocytochrome c by SegmentCoupling, Int. J. Pept. Protein Res. 27:191-200 (1986); and H. Hojo etal., Protein Synthesis using S-Alkyl Thioester of Partially ProtectedPeptide Segments, Synthesis of DNA-Binding Protein of Bacillusstearothermophilus, Bull. Chem. Soc. Jpn. 65:3055-3063 (1992). However,this method still requires the use of protecting groups on all Lysineside chain amino groups, selective N-α protection of one or moresegments, and laborious purification steps, involving purification,reprotection, and repurification.

The use of multiply protected peptide segments is incompatible with theoverall scheme of engineering proteins using peptides produced by meansof recombinant DNA expression as a source. Protected peptide segmentmethods are labor-intensive, and the protected peptide segments haveunpredictable handling properties, partly due to the solubility andligating difficulties of protected peptide segments. Often, largeprotected peptide segments are minimally soluble in even the mostpowerful polar aprotic solvents such as dimethylsulfoxide (DMSO) anddimethylformamide (DMF). The problem of insolubility in protectedpeptide segments has been addressed with limited success in severalways, including the use of (1) partial protecting group strategy whichmasks all side chains except those of Ser, Thr, and Tyr; (2) minimalprotecting group strategy that masks only thiol and amino side chains;and (3) using reversible protection of a backbone amide moiety toprevent aggregation/insolubility. Protecting groups used in the latterapproach alter peptide conformations. Use of backbone protecting groupsis not yet straightforward or predictable and requires significantexperimentation for each target polypeptide chain.

There are a number of techniques for ligating unprotected peptidesegments via unnatural backbone linkages. In contrast, there are fewmethods for achieving a “native chemical ligation.” A “native chemicalligation” is the chemoselective reaction of unprotected or N-terminalCysteine protected peptide segments with another unprotected peptidesegment resulting in the formation of a ligated peptide with an amidebond at the ligation site. The fully assembled target polypeptides ofthe invention comprise one, two or more native chemical ligation sites.

Accordingly, there is a need in the art for rapid methods ofsynthesizing assembled polypeptides via chemical ligation of two or moreunprotected peptide segments using a solid support, with improved yieldsand facilitated handling of intermediate products.

The present invention makes possible, inter alia, the rapid solid-phasesynthesis of large polypeptides with a natural peptide backbone vianative chemical ligation of two or more unprotected peptide segmentswhere none of the reactive functionalities on the peptide segments needto be temporarily masked by a protecting group. The present inventionaccomplishes for the first time, solid phase sequential chemicalligation of peptide segments in an N-terminus to C-terminus direction,with the first solid phase-bound unprotected peptide segment bearing aC-terminal α-thioester that reacts with another unprotected peptidesegment containing an N-terminal Cysteine and a C-terminal thioacid.

Other embodiments of the invention also permit solid-phase nativechemical ligation in the C- to N-terminus direction, with temporaryprotection of N-terminal cysteine residues on an incoming (second)peptide segment. Those of ordinary skill in the art will readilyappreciate that the invention may also include the use of nonnativechemical ligation to sequentially ligate peptide segments via unnaturallinkages on a solid phase. Alternatively, the invention may include theuse of native chemical ligation of peptide segments wherein said peptidesegments comprise one or more unnatural backbone linkages.

REFERENCES

Matthys J. Janssen, “Thiolo, Thiono, and Dithio Acids and Esters,”Chptr. 15 of The Chemistry of Carboxylic Acids and Their Esters (1969).

Schnolzer et al., Science 256:221-225 (1992)

Rose et al. J. Am Chem. Soc. 116:30-34 (1994)

Liu et al., Proc. Natl. Acad. Sci. USA 91:6584-6588 (1994).

Dawson et al. Science 266:77-779 (1994).

PCT/US95/05668, WO 96/34878

Sakakibara S., Biopolymers (Peptide Science), 37:17-28 (1995).

Tam et al., PNAS USA, 92:12485-12489 (1995).

SUMMARY OF THE INVENTION

The present invention provides, inter alia, novel methods of producinglarge polypeptides by native chemical ligation of peptide segments inaqueous solution to an unprotected solid phase bound peptide withoutneed for protecting groups on the peptide segments, or, with temporaryprotection of the N-terminal cysteine of incoming peptide segments.Among the many advantages of this embodiment of the invention are: easeof purification of the intermediate and final products; faster ligationreactions; rapid synthesis of large polypeptides with a natural peptidebackbone; ease of ligating reactions due to the lack of protectinggroups and resultant enhanced solubility of peptide segments in aqueousor mixed aqueous/organic solutions; chemoselective ligation due to thelack of reactivity of the thioester moiety with other functional groupspresent in both reactive peptide segments to form stable co-products,resulting in a purer final product without side reactions; adaptabilityto monitoring on the solid phase via MALDI mass spectrometry or ESI MS(electrospray ionization mass spectrometry); decreased racemization dueto the use of mild activation using a thioester and the avoidance ofelevated pHs; the polypeptide product is obtained directly inunprotected form; and adaptability to automation and combinatorialtechniques.

A significant advantage of the solid phase ligations over solutionligations is that the solid phase ligation methods do not requirearduous HPLC (high pressure liquid chromatography) purification andlyophilization steps after each ligating reaction, whereas ligations insolution do. Thus, the solid phase ligations eliminate manytime-consuming purification steps that decrease the recovery of finalproduct. Instead, the solid phase sequential ligation methods heredescribed only require a single HPLC purification and lyophilizationstep after the final unprotected peptide segment has been ligated andthe assembled peptide is cleaved from the solid phase. The eliminationof these time-consuming purification steps allows for faster synthesisof the final product, i.e. the assembled peptide, than would theanalogous route in solution. Ready purification of the desired solidphase-bound product from soluble coproducts presents a tremendousadvance in terms of the yield of the ultimate assembled polypeptide.

Another advantage of solid phase ligations is that they permit higherconcentrations of reactants which leads to faster reaction rates. Forexample, by using an excess at high concentration of the incomingpeptide segment as compared to the solid phase-bound peptide, reactionscan reach completion much faster. The excess peptide segment can readilybe washed off the solid phase after the ligation reaction is complete.Increased yields of final product can be accomplished by increasingconcentrations of peptide segments. For example, the solid phase-boundpolypeptide can be dried out on the solid-phase and ressolvated inligation solution. Alternatively, the solid phase-bound peptide can bewashed with a solution of incoming peptide segments at highconcentration.

Other advantages of the present invention are that it allows forsynthesis of much larger peptides and proteins than are presentlyattainable by conventional methods, it is amenable to automation, andthe use of high resin loadings allow for easy scale up. Moreover,ligation in the N- to C-terminal direction permits the use of crudepeptide segments without need for purification or lyophilization, sincetermination products formed during stepwise solid phase synthesis of thepeptide segments will be unreactive with the solid phase-bound peptide.

In one embodiment, the invention comprises a method of producing anassembled peptide having a native peptide backbone by ligating peptidesegments in the N- to C-terminal direction, comprising: a) covalentlybinding an unprotected first peptide segment to a solid phase via alinker comprising a cleavable moiety, wherein said cleavable moiety isstable under ligation conditions and said unprotected first peptidesegment is bound to said cleavable moiety at its N-terminus and has anα-thioester at its C-terminus; b) optionally introducing a secondunprotected peptide segment, wherein said second segment comprises acysteine residue at its N-terminus and a thioacid at its C-terminus,under conditions suitable to permit ligation between said firstunprotected peptide segment and said second unprotected peptide segmentto form a natively ligated peptide bound to said solid phase, whereinsaid solid phase-bound peptide comprises a thioacid at its C-terminus,and subsequently converting said solid phase-bound peptide thioacid to athioester; (c) optionally repeating step (b) with additional unprotectedpeptide segments; (d) introducing a final unprotected peptide segment,comprising a cysteine residue at its N-terminus, under conditionssuitable to permit ligation between said solid phase-bound peptide andsaid final unprotected peptide segment. In a preferred embodiment, thecleavable moiety is cleaved to release the solid phase-bound peptide inthe form of the assembled peptide. In another preferred embodiment,cleavable moiety is a cleavable linker capable of being cleaved forpurposes of monitoring the sequential ligation reactions. In anotherembodiment, the first unprotected peptide segment is added as apeptide-αCOSH thioacid and subsequently converted to a thioester.

The sequential ligation in the N- to C-terminus direction is asurprisingly effective and elegant means of obtaining chemoselectiveligation of unprotected peptide segments without racemization. Beforethe present invention, sequential ligations were not conducted in the N-to C-terminal direction due to concerns regarding racemization at theαCOX at the C-terminus of the peptide (peptide-αCOX). Using the presentinvention, the αCOSH at the C-terminus of the peptide segment is mildlyactivated to a thioester and the ligating reaction is carried out in theabsence of base, in an aqueous buffered solution, resulting in mildconditions that do not generate racemic mixtures.

The methods of the invention can be used for native chemical ligation ofpeptide segments produced by stepwise solid phase synthesis. The lastpeptide segment to be added at the C-terminal end of the last solidphase-bound peptide in the reaction scheme may be a recombinantlyexpressed peptide having an N-terminal Cysteine residue (Cys-recombinantpeptide). The thioacid moiety, which is activated to a thioester moiety,can be placed anywhere a native chemical ligation is desired, includingon a side chain. Thus, the sequential ligations of the invention are notlimited to linearly assembled peptides.

In another embodiment, there is the use of unprotected peptide segmentmiddle pieces each having an N-terminal cysteine residue thatparticipate in native chemical ligation.

In another embodiment, the invention comprises a method of producing anassembled peptide having a native peptide backbone by ligating peptidesegments in the C- to N-terminal direction, comprising: a) covalentlybinding an unprotected first peptide segment to a solid phase via acleavable handle comprising a cleavable moiety, wherein said cleavablemoiety is stable under ligation conditions and said unprotected firstpeptide segment is bound to said cleavable moiety at its C-terminus andhas a Cysteine at its N-terminus; b) introducing a second peptidesegment, wherein said second segment comprises a cysteine residue at itsN-terminus and an alpha-thioester at its C-terminus, and wherein saidsecond peptide segment has a protecting group bound to its N-terminalcysteine residue, under conditions suitable to permit ligation betweensaid first peptide segment and said second N-terminally protectedpeptide segment to form a natively ligated peptide bound to said solidphase, wherein said solid phase-bound peptide comprises a protectinggroup bound to an N-terminal cysteine; c) removing said protecting groupfrom solid phase-bound peptide; (d) optionally repeating steps b) and c)with additional peptide segments comprising an N-terminal Cysteine and aC-terminal alpha thioester, wherein said additional peptide segmentshave a protecting group bound to their N-terminal cysteine residue (e)introducing a final peptide segment, comprising an alpha-thioester atits C-terminus, providing that if said final peptide segment comprisesan N-terminal Cysteine, said N-terminal Cysteine is protected by aprotecting group, wherein said introducing occurs under conditionssuitable to permit ligation between said solid phase-bound peptide andsaid final peptide segment; and (e) optionally removing said protectinggroup from the N-terminal cysteine of said solid phase-bound peptide.

In another embodiment, the invention comprises a method of producing anassembled peptide having a native peptide backbone by ligating peptidesegments in the C- to N-terminal direction, comprising: a) covalentlybinding an unprotected first peptide segment to a solid phase via acleavable handle comprising a cleavable moiety, wherein said cleavablemoiety is stable under ligation conditions and said unprotected firstpeptide segment is bound to said cleavable moiety at its C-terminus andhas a Cysteine at its N-terminus; b) optionally introducing a secondpeptide segment, wherein said second segment comprises a cysteineresidue at its N-terminus and an alpha-thioester at its C-terminus, andwherein said second peptide segment has a protecting group bound to itsN-terminal cysteine residue, under conditions suitable to permitligation between said first peptide segment and said second N-terminallyprotected peptide segment to form a natively ligated peptide bound tosaid solid phase, wherein said solid phase-bound peptide comprises aprotecting group bound to an N-terminal cysteine, and subsequentlyremoving said protecting group from solid phase-bound peptide; (c)optionally repeating step (b) with additional peptide segmentscomprising an N-terminal Cysteine and a C-terminal alpha thioester,wherein said additional peptide segments have a protecting group boundto their N-terminal cysteine residue; (d) introducing a final peptidesegment, comprising an alpha-thioester at its C-terminus, providing thatif said final peptide segment comprises an N-terminal Cysteine, saidN-terminal Cysteine is protected by a protecting group, wherein saidintroducing occurs under conditions suitable to permit ligation betweensaid solid phase-bound peptide and said final peptide segment; and (e)optionally removing said protecting group from the N-terminal cysteineof said solid phase-bound peptide.

In yet another embodiment, there is the solid phase sequential ligationof peptide segments in either or both directions, using a cleavablelinker to monitor the ligation reactions via mass spectrometry and topurify the assembled peptide from the solid phase.

Another embodiment is a method of bidirectional solid phase nativechemical ligation, comprising providing a first peptide segment bound toa solid support via one of its internal amino acid residues, whereinsaid first peptide segment comprises an N-terminal Cysteine and aC-terminal thioester, and ligating a second peptide segment to eitherterminus.

In another embodiment, there is provided a kit comprising an unprotectedpeptide segment, covalently bound via an internal amino acid side chainfunctional group to a cleavable handle, wherein said cleavable handle islinked to a solid phase via a chemoselective functional groupcomplementary to a chemoselective functional group on the solid phase.Said kit can be used for solid phase chemical ligation of unprotected orN-terminal cysteine-protected peptide segments to the solid phase-boundpeptide. A preferred example of such a cleavable handle is afunctionality cleavable handle, X-aminoethylsulfonylethyloxycarbonyl(wherein X═CH₃COCH₂CH₂CH₂CONHCH₂—MSC— or X=AOA—NHCH₂—MSC—.(AOA=aminooxyacetal).

In another embodiment, there are methods of using bromoacetic acid oriodoacetic acid to convert a peptide segment thioacid (peptide-αCOSH) toa thioester (peptide-aCOSR), on a solid phase.

In yet another embodiment, there is provided a method of monitoring thesolid phase sequential ligation process on the solid phase via MALDI orESI mass spectrometry, using cleavable linkers. Monitoring via ESI MScan also be accomplished using a TFA-cleavable linker or, when MALDI isthe mass spectrometric method used, a photocleavable linker maypreferably be used.

In a further embodiment, there are provided novel methods of preparingmodular large peptide or protein libraries using combinations of theaspects of the invention described herein. Particularly useful are themethods of solid phase sequential ligation of peptide segments torapidly synthesize multiple analogs of known proteins or polypeptides.

Kits and apparatus for assembling polypeptides and polypeptide librariesby the processes described herein are also provided.

One of skill in the art will readily appreciate that each of theembodiments of the invention can be combined with other embodiments toobtain a wide range of useful inventions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of a solid phase native chemical ligationscheme, in the N- to C-terminus direction. In one embodiment, the linkeris an MSC handle, which is cleavable yet stable under ligationconditions. In another embodiment, the unprotected first peptide segmentis covalently bound to a solid phase (resin) via an aminooxy-ketonelinkage.

FIG. 2A illustrates the stability of a 13-residue peptide-α-COSH (aminoacids 36-48; SEQ ID NO:2) with a Cysteine residue at the N-terminusunder ligation conditions. The HPLC chromatogram shows that only a smallpercentage of the peptide cyclized or formed larger aggregates, evenafter overnight storage under ligation conditions.

FIG. 2B illustrates the stability of the same 13-residue peptide-αCOSH(amino acids 36-48; SEQ ID NO:2) in the presence of a thioester peptidehaving a molecular weight of 1230.2. The HPLC chromatogram shows thatthe Cys-α-COSH peptide is adequately stable to use in ligation withoutsignificant reaction with itself. Furthermore, such byproducts as areformed in small proportion by reaction of the 13-residue peptide-αCOSH(having an N-terminal cysteine) with itself are unreactive with aresin-bound peptide α-COSR and are readily removed by simple filtrationand washing.

FIGS. 3A, 3B and 3C show HPLC chromatograms of the effect of hydrazineon the removal of the MSC handle from a peptide having an N-terminalCysteine residue (amino acids 20-35; SEQ ID NO:2). The peak correlatingwith the mass of 1708.2 represents the desired peptide (amino acids20-35; SEQ ID NO:2) with the MSC handle removed. The peak correspondingto the mass of 1814.5 represents a reactive side product formed uponcleavage that can react with the desired peptide without the MSC handle.FIG. 3A shows a fairly large peak at the 1814.5 mw when an aliquot ofthe peptide was placed in 6M guanidine.HCl, 0.1 M NaPi, pH 7.5, thendiluted into 1 N NaOH for 2 min., then quenched with 1 N HCl. FIG. 3B isan HPLC chromatogram of the resulting product when the conditions ofFIG. 3A are repeated with the inclusion of 50 mM hydrazine in the 6 Mguanidine.HCl solution. FIG. 3C is an HPLC chromatogram of the resultingproduct when the conditions of FIG. 3A are repeated with 200 mMhydrazine in the 6 M guanidine.HCl solution. Hydrazine scavenges theside product, resulting in a purer product.

FIG. 4 is an HPLC chromatogram of the removal of a cleavable MSC handlefrom a peptide that does not have an N-terminal Cysteine residue, butrather an N-terminal Leucine residue and a Cysteine residue in itsapproximate center. The molecular weight of the peptide with the MSChandle is 4022.4 (amino acids 1-35; SEQ ID NO:2) and without the MSChandle, 3745.1 (amino acids 1-35; SEQ ID NO:2). An aliquot of thepeptide in 6M guanidine.HCl, 0.1M NaAc, pH 4.6 was diluted into 6Mguanidine.HCl, 0.1M NaAc, pH 14 for 2 min., quenched with 6Mguanidine.HCl, 0.1M NaAc, pH 2.0. The HPLC shows that an internalreaction with the side product still occurs, to form the peak having amw of 3979.7 (corresponding to the modification by theLEV-NHCH₂-handle), but that the extent of the reaction is less than thatoccurring with a peptide having an N-terminal Cysteine.

FIG. 5A is a reaction scheme showing the preparation of the PEGA resinused as the solid support in N- to C-terminal sequential ligations.Steps A and B 1 are optional steps to produce a photolabile linker foruse with MALDI analysis of the resin samples.

FIG. 5B is a diagram illustrating a generalized scheme for preparing asolid phase (resin) for use in the solid phase sequential ligations ofthe invention. Structure 1 is a cleavable linker useful for monitoringthe progress of coupling and ligation reactions by mass spectrometry.For example, a photo-cleavable linker can be used for on-resinmonitoring by MALDI MS, whereas a TFA cleavable linker can be used formonitoring by electrospray MS. Once structure 1 is coupled to the resin,the protecting group (PG) is removed and a functional moiety (structure3) capable of chemoselective reaction with the first peptide segment, isadded to the resin. Once 3 is coupled to the resin, the protecting groupis removed to give structure 4, which is ready for chemoselectivereaction with structure 5, a peptide modified with a cleavable handleand a functional group capable of reaction with the now modified resin(4). Once all subsequent ligtions are complete, the “cleavable handle”is cleaved to release the full length peptide (assembled peptide) fromthe solid phase.

FIG. 6 is a reaction scheme illustrating the derivatization of PeptideSegment 1 (the N-terminal peptide segment) (amino acids 1-35; SEQ IDNO:2).

FIGS. 7A and 7B are HPLC chromatograms of the coupling of a firstunprotected peptide segment (1) (amino acids 1-35; SEQ ID NO:2) to thesolid support, in this example, an AOA-functionalized resin (PEGA). FIG.7A is an HPLC of the peptide solution as added to the resin. FIG. 7B isan HPLC of the supernatant after reaction of the peptide (amino acids1-35; SEQ ID NO:2) with a molar excess of the resin overnight. Asignificant amount of the peptide has been removed from the supernatant,indicating that it has been bound to the resin after the overnightreaction.

FIGS. 8A and 8B are HPLC chromatograms of the same experiments reflectedin FIGS. 7A and 7B, except with Isco resin beads as the solid phase(amino acids 1-35; SEQ ID NO:2).

FIGS. 9A, 9B, and 9C are analyses of the products after step 1 of thisfigure, binding of the first unprotected peptide segment to the solidphase. FIG. 9A is an analytical HPLC chromatogram of the (base plushydrazine) cleavage of the resin-bound peptide. FIG. 9B is a MALDI massspectrum of the resin, showing a peak corresponding to (1), theresin-bound peptide. FIG. 9C is a MALDI mass spectrum after basecleavage of the linker, showing the lack of a peak corresponding to (1),and showing that no peptide is sticking to the solid phase (resin).

FIGS. 10A, 10B, and 10C are analyses of the products after step 3 ofthis figure, i.e., ligating of the second unprotected peptide segment(2) (amino acids 36-48; SEQ ID NO:2) to the resin-bound peptide (1)(amino acids 1-35; SEQ ID NO:2). FIG. 10A is an analytical HPLC of theproduct (amino acids 1-48; SEQ ID NO:2), resin-bound peptideintermediate, showing a large peak with mass of (1)+(2). FIG. 10B is aMALDI mass spectrum of the resin before cleavage of the linker, and FIG.10C is a MALDI mass spectrum of the resin after base cleavage of thelinker.

FIG. 11 is an HPLC chromatograph of the desalted, lyophilized peptideproduct (1+2+3 of Table 1) after 2 sequential ligations on a solid phase(Isco resin) in the N- to C-terminal direction. The tallest peakcorresponds to the crude, lyophilized product, indicating approximately36% yield.

FIGS. 12A and 12B are ESI MS (electrospray ionization mass spectra) ofthe main peak corresponding to the assembled peptide (1+2+3 of Table 1).FIG. 12B is a reconstructed display of the mass spectrum of FIG. 12A,showing the mass of the product ligated peptide.

FIG. 13 is an HPLC chromatogram of the desalted, lyophilized peptide(1+2+3) after base cleavage of the linker to remove the assembledpeptide from the solid phase (PEGA resin).

FIGS. 14A and 14B are electrospray ionization mass spectra of the 7434mass peak, wherein FIG. 14B is a reconstruction of the mass spectrum ofFIG. 14A.

FIGS. 15A, 15B and 15C are 3 HPLC chromatograms illustrating that thesolid support technique can be used for both purification and ligation.FIGS. 15A and 15B show solution processing of a crude peptide before andafter removal of DNP groups, respectively. Both HPLCs show a crudemixture of peptides. FIG. 15C is an HPLC chromatogram of the samepeptide solution shown in FIG. 15A, after coupling to a solid support,removal of DNP groups and base cleavage from the solid phase, resultingin a significantly purer assembled peptide product.

FIGS. 16A and 16B illustrate the reaction scheme for synthesis ofMIF(1-115) (SEQ ID NO:4) via solid phase sequential native ligations inthe N-terminal to C-terminal direction using MIF 1-59 thioester as astarting material (amino acids 1-59; SEQ ID NO:4) and having MIF 1-80thioacid as an intermediate (amino acids 1-80; SEQ ID NO:4).

FIG. 17A is a reaction scheme for the modification of the N-terminalpeptide segment. FIG. 17B is a diagram illustrating the modification ofthe aqueous-compatible solid phase in preparation for coupling the firstunprotected peptide segment.

FIG. 18A is a reaction scheme for the coupling of N-terminal modifiedMIF(1-59) (amino acids 1-59; SEQ ID NO:4) to a solid phase. FIG. 18B isan HPLC chromatogram of the released peptide after base cleavage, havingan expected mass of 6271 Da. FIGS. 18C and 18D are electrospray massspectra of the main component of the released peptide after cleavage ofthe cleavable handle. FIG. 18D is a reconstruction of FIG. 18C.

FIG. 19A is a diagram of the ligation step to form resin-bound MIF(1-80)(amino acids 1-80; SEQ ID NO:4). FIG. 19B is an HPLC chromatogram of theproducts after cleavage of the cleavable handle. FIGS. 19C and 19D aremass spectra of the main components of the released peptide after basecleavage, having an expected mass of 8502 Da. FIG. 19D is areconstructed display of the mass spectrum of FIG. 19C.

FIG. 20A is a diagram of the ligation step to form resin-boundMIF(1-115) (SEQ ID NO:4). FIG. 20B is an HPLC chromatogram of theproducts after cleavage of the cleavable handle. FIG. 20C and FIG. 20Dare mass spectra of the released products after base cleavage, having anexpected mass of 12450 Da.

FIG. 21 is a schematic diagram of solid phase ligations in the C- toN-terminus direction. The “resin” represents a solid phase. The triangleand its sideways M-shaped partner are complementary functional groupsthat chemoselectively form a covalent bond. The “handle” is a cleavablehandle that can be cleaved to remove the assembled peptide product fromthe solid phase. The undulating lines comprise amino acid residues ofpeptide segments. The “PG” represents a protecting group, which can beplaced either on a side chain thiol or on the α-amino group of theN-terminal cysteine. Steps 2 and 3 can be repeated, as indicated by thearrow marked 4, for additional peptide segments. Also, a cleavablelinker for purposes of monitoring the coupling and ligating reactionscan be added between the “handle” and the “resin.”

FIG. 22 is a reaction scheme for solid phase sequential ligation in theC- to N-terminal direction of PLA2G5.

FIG. 23 is a reaction scheme for synthesizing a Cam ester derivative forsolid phase sequential ligation in the C- to N-terminal direction.

FIG. 24 is a reaction scheme for synthesizing the C-terminal peptidesegment for solid phase sequential ligation in the C- to N-terminaldirection.

FIGS. 25A, B, and C is a diagram of a scheme for synthesizing anassembled polypeptide via bidirectional solid phase sequential ligationof two or more peptide segments.

FIG. 26 are HPLC chromatographs following the solid phase solid phasenative chemical ligation of 3 peptide segments in the N- to C-terminaldirection, resulting in the assembled peptide, C5a 1-74 (SEQ ID NO:3).

FIG. 27 is a reaction scheme for synthesis of a C-terminal peptidesegment for use in the solid phase native chemical ligations describedherein, using a CAM ester cleavable handle to remove the synthesizedpeptide segment from the solid phase.

FIG. 28 is the HPLC chromatograph and reconstructed ESI MS of theassembled peptide resulting from solid phase sequential ligation of 3peptide segments: peptide segment 1 (CADRKNILA) (amino acids 19-27; SEQID NO:1), peptide segment 2 (CYGRLEEKG) (amino acids 10-18; SEQ ID NO:1)and peptide segment 3 (ALTKYGFYG) on solid phase in the C- to N-terminaldirection, using Fmoc protecting groups to yield the peptide product(SEQ ID NO:1).

FIG. 29A is the HPLC chromatograph and ESI MS of the final ligationproduct, i.e. the first ligation product (SEQ ID NO:1) ligated to thethird peptide segment (ALTKYGFYG) (amino acids 1-9; SEQ ID NO:1),resulting from solid phase sequential ligation of 3 peptide segments inthe C- to N-terminal direction, using ACM as the protecting group.

FIGS. 30A and B are HPLC chromatographs and reconstructed ESI MS of thesteps of synthesizing Phospholipase A2 Group 5, a 118 residue protein(SEQ ID NO:5), using solid phase sequential native chemical ligation offour peptide segments in the C- to N-terminal direction. The firstpeptide segment is PLA2G5 88-118; the second is PLA2G5 59-87, the thirdis PLA2G5 26-58, and the fourth is PLA2G5 1-25. FIGS. 30A and B are anHPLC chromatograph and reconstructed ESI MS of the first peptidesegment, respectively. FIGS. 30A and B are an HPLC chromatograph andreconstructed ESI MS, respectively, of the ligation product of the firstand second peptide segments (PLA2G5 59-118). FIGS. 30A and B are an anHPLC chromatograph and reconstructed ESI MS, respectively, of PLA2G526-118, the ligation product of PLA2G5 59-118 and PLA2G5 26-58 (thethird peptide segment). FIG. 30A and B are HPLC chromatograph andreconstructed ESI MS, respectively, of PLA2G5 1-118, the assembledpolypeptide.

DESCRIPTION OF SPECIFIC EMBODIMENTS

Terminology

Amino acids: Amino acids include the 20 genetically coded amino acids,rare or unusual amino acids that are found in nature, and any of thenon-naturally occurring and modified amino acids.

Aqueous solution: solutions containing water, including up to 8M urea inwater, up to 6M guanidine.HCl in water, up to 60% acetonitrile in water.

Assembled Peptide: the final product of a solid phase sequential orbidirectional ligation, after cleavage of the cleavable handle. Theassembled peptide comprises at least two separate peptide segmentssequentially ligated on a solid phase. The assembled peptide may or maynot have biological activity.

Cleavable Handle: A cleavable moiety that is capable of beingselectively cleaved to release the assembled peptide from the solidphase. The cleavable handle must be capable of resisting cleavage underconditions suitable for coupling, activating, deprotecting, ligating,washing, and other steps involved in the formation of an assembledpeptide. The cleavable handle must also be stable to conditions used toproduce the first peptide segment that is capable of being bound to asolid phase, including, for example, stepwise solid phase peptidesynthesis. The cleavable handle preferably is located directly adjacentto the first peptide segment such that upon cleavage of the cleavablehandle, the desired assembled peptide is released from the solid phase.The cleavable handle may be selected from any of the variety ofcleavable handles used by those in the field. See, e.g., L. Canne etal., Tetrahedron Letters, 38(19):3361-3364 (1997); Ball et al., J. Pept.Sci, 1:288-294 (1995); Funakoshi et al, PNAS USA, 88:6981-6985 (1991);Funakoshi et al., J. Chromatog. 638:21-27 (1995); Garcia-Echeverria etal., J. Chem. Soc., Chem. Commun., 779-780 (1995). A preferred cleavablehandle is Boc-HN—CH2-CH2-SO2-CH2-CH2-O—CO—ONp (Boc-HNCH2-MSC-) or afunctionalized cleavable handle, X-aminoethylsulfonylethyloxycarbonyl(wherein X=CH3COCH2CH2CH2CONHCH2-MSC- or X=AOA-NHCH2-MSC-).(AOA=aminooxyacetal). Another preferred cleavable handle is a CAM ester.See Ceccato, M. L. et al., Tetrahedron Lett. 31:6189-6192 (1990).

Cleavable Linker: A cleavable moiety that is capable of beingselectively cleaved to monitor the solid phase sequential ligation usingmass spectrometry of small samples of the reaction mixture at any pointduring the ligation procedure, i.e. after ligating of the second peptidesegment, after ligating of the third peptide segment, and so forth. Thecleavable linker must be stable under coupling and ligating conditions,deprotecting conditions (if needed), and washing conditions. Preferredcleavable linkers include photolabile linkers and TFA-labile linkers.

Coupling: Chemoselective reactions involving covalent binding of a firstpeptide segment to a solid phase.

Ligating: Chemoselective reactions involving covalent binding of apeptide segment to a solid phase-bound peptide.

Linker: A covalent linkage linking various moieties. For example, alinker may link a first peptide segment and a solid support, and such alinker may optionally comprises any number of moieties, including acleavable handle, a cleavable linker, complementary functional groupscapable of chemoselectively forming a covalent bond (e.g., amino-oxy andketone to form an oxime).

Peptide: A polymer of at least two monomers, wherein the monomers areamino acids, sometimes referred to as amino acid residues, which arejoined together via an amide bond. For purposes of this invention, theterms “peptide,” “polypeptide,” and “protein,” are largelyinterchangeable as all three types can be made via the methods describedherein. Peptides are alternatively referred to as polypeptides. Aminoacids include the L and D isoforms of chiral amino acids.

Peptide segment: A peptide or polypeptide, having either a completelynative amide backbone or an unnatural backbone or a mixture thereof,ranging in size from 2 to 1000 amino acid residues, preferably from 2-99amino acid residues, more preferably from 10-60 amino acid residues, andmost preferably from 20-40 amino acid residues. Each peptide segment cancomprise native amide bonds or any of the known unnatural peptidebackbones or a mixture thereof. Each peptide segment can be prepared byany known synthetic methods, including solution synthesis, stepwisesolid phase synthesis, segment condensation, and convergentcondensation. The final peptide segment to be added to form theassembled peptide product can be recombinantly expressed.

Protecting Group: A chemical moiety capable of protecting a functionalgroup from reacting with another functional group, and removable withoutdamage to the formed amino acid or peptide.

Sequential ligation: ligating three or more peptide segments together inorder from C-terminus to N-terminus or from the N-terminus toC-terminus, depending on the directionality chosen, to obtain anassembled peptide product. The directionality of the sequentialligations will always start from the solid phase-bound first peptidesegment to the last peptide segment to be added to form the assembledpeptide product.

Solid Phase: A material having a surface and which is substantiallyinsoluble when exposed to organic or aqueous solutions used forcoupling, deprotecting, and cleavage reactions. Examples of solid phasematerials include glass, polymers and resins, including polyacrylamide,PEG, polystyrene PEG-A, PEG-polystyrene, macroporous, POROS™, cellulose,reconstituted cellulose (e.g. Perloza), nitrocellulose, nylon membranes,controlled-pore glass beads, acrylamide gels, polystyrene, activateddextran, agarose, polyethylene, functionalized plastics, glass, silicon,aluminum, steel, iron, copper, nickel and gold. Such materials may be inthe form of a plate, sheet, petri dish, beads, pellets, disks, or otherconvenient forms. Sheets of cellulose can be used as a solid phase inthe present invention to accomplish spot ligation in a spatiallyaddressable array. Many of the examples and embodiments described hereinrefer to resins, which are a type of solid phase, and one of ordinaryskill in the art would understand that such examples are not meant to belimited to resins, but to solid phases in general. The terms solid phaseand solid support are used herein interchangeably.

Solid Phase-bound Peptide: a solid phase-bound peptide comprises atleast one peptide segment bound to a solid phase via any variety ofcleavable linkers, handles or moieties. A solid phase-bound peptide caninclude any of the intermediate peptide products of the sequentialligation reactions, including the final solid-phase bound peptideproduced after the final peptide segment is ligated to the penultimatesolid phase-bound peptide.

Thioacid: An ionizable thioacid moiety, represented by either —COSH or—COS⁻, often referring to a peptide thioacid, represented by “peptideα-COSH” or “peptide α-COS⁻.”

Thioester: A moiety represented by -COSR, often connected to a peptide.For example, a peptide thioester may be represented as “peptide α-COSR”.The R group may be any number of groups, including 1-15 C functionalizedalkyl, straight or branched, 1-15 C aromatic structures, 1-4 amino acidsor derivatives thereof, preferably wherein the R group is selected suchthat the peptide-alpha-COSR is an activated thioester. In a preferredembodiment, R=—CH3-Ø, -Ø. The term “thioester” is commonly used, but thetrue IUPAC term is “thioloester.” See Matthys J. Janssen, supra

I. Solid Phase Sequential Native Ligation of Unprotected PeptideSegments in the N- to C-Terminal Direction

There have been few reports of proteins synthesized by sequential,multiple ligations of three or more unprotected peptide segments. Suchsequential ligations of free peptide segments in solution consequentlyrequire a purification (e.g. HPLC) after each ligation and typicallyrequire temporary protection of one of the functionalities of the middlesegments.

One aspect of the present invention is a solid phase sequential ligationtechnique which avoids the need for multiple purifications and the needto temporarily protect the middle peptide segments. This strategyemploys (1) the modification of the N-terminal peptide segment with acleavable handle functionalized with a group capable of chemoselectivereaction with the solid support and (2) sequential native chemicalligations of unprotected peptide segments in an N- to C-terminaldirection. Native chemical ligation involves reaction of an unprotectedpeptide segment bearing a C-terminal α-thioester with a secondunprotected peptide segment containing an N-terminal Cysteine residue.Thiol exchange yields a thioester-linked intermediate whichspontaneously rearranges to a native amide bond at the ligation site. Wehave determined that a peptide segment bearing an N-terminal Cysteineand a C-terminal thioacid is sufficiently stable under native ligationconditions that it requires no temporary protection of the C-terminalthioacid functionality. Accordingly, these peptide segments can be usedas the middle segments in a sequential ligation scheme involving threeor more peptide segments as shown in FIG. 1. Once such a middle segmenthas ligated to the solid phase-bound thioester-containing peptide togenerate a solid phase-bound peptide thioacid, the thioacid is easilyconverted to a thioester and can be reacted with the N-terminal Cysteineof the next peptide segment to be ligated. Alternatively, the incomingpeptide segment may have an internal amino acid with a nonnatural sidechain bearing amino and thiol moieties on adjacent c atoms, i.e. in a1,2 relation to one another, and an unreactive, unprotected non-cysteineamino acid residue at its N-terminus, which would lead to a nonlinearassembled peptide. Multiple ligations of distinct peptide segments toform an assembled peptide bound to the solid phase are contemplated.Once all ligations are complete, the linker binding the solidphase-bound peptide to the solid phase is cleaved, releasing theassembled peptide, i.e., the full length peptide. This technique isapplied to the total chemical synthesis of a random peptide ofartificial sequence (Table 1 in Examples Section), and human MacrophageMigration Inhibitory Factor (MIF), a 115 amino acid cytokine involved inimmune system function. See FIGS. 16-20.

A. Peptide Synthesis

Peptide segments were synthesized in stepwise fashion by establishedmachine-assisted solid-phase methods on polystyrene resins using in situneutralization/HBTU activation protocols for Boc chemistry (L. Canne etal., Tetrahedron Lett. 38:3361-3364 (1997)) on Boc-aminoacyl-OCH₂-PAMresins, thioester-generating resins (Hojo, et al., Bull. Chem. Soc. Jpn.64:111-117 (1991)), or thioacid-generating resins. After chain assemblywas complete, peptides were deprotected and simultaneously cleaved fromthe resin by treatment with anhydrous HF containing 5% p-cresol,lyophilized, and purified by preparative HPLC. The N-terminal peptidesegment was modified prior to HF cleavage as outlined in FIG. 17A.

B. Preparation of the Solid Phase

The solid phase is prepared as depicted in FIGS. 5A and 5B. FIG. 5A is ascheme for preparing PEGA resin as a solid support. FIG. 5B is ageneralized diagram for the preparation of any solid phase. Anamino-Spherilose™ (Isco) affinity resin was derivatized withBoc-aminooxyacetic acid as shown in FIG. 17B.

Other resins to be used as the solid phase include EAH Sepharose(Pharmacia), Amino PEGA (Novabiochem), CLEAR base resin (PeptidesInternational), long chain alkylamine controlled pore glass (Sigma),HCl.PEG polystyrene (PerSeptive Biosystems), Lysine Hyper D resin(Biosepra), ArgoGel Base resin (Argonaut Technologies). These resins areavailable in amino-derivatized form or are readily converted toamino-derivatized form.

C. Coupling of Modified N-terminal Peptide Segment to Solid Phase

The modified peptide, containing a ketone moiety, as depicted in FIG.17A, is dissolved in 6M guanidine.HCl, 0.1M Na acetate, 0. 15Mmethionine, pH 4.6 (1.6 mM) and added to the aminooxy functionalizedsolid support, which had previously been thoroughly washed in the samebuffer, and allowed to react at room temperature overnight (FIG. 16A,Step #1).

II. Ligation in the N- to C-Terminal Direction

A. Ligation Reactions

The peptide segment to be ligated to the resin-bound peptide thioesterwas dissolved in 6M guanidine.HCl, 0.1M Na acetate, 0.15M methionine,0.5% thiophenol, pH 7.5 (3.7-4.0 mM) and added to the resin boundpeptide thioester, which was thoroughly washed in the same buffer, andallowed to react at room temperature overnight (FIGS. 16A and 16B, Steps#2 and 4). Preferably the concentration of the first peptide segment canrange from 1 to 150 mM; more preferably from 5-100 mM, most preferablyfrom 10-50 mM, depending on the particular peptide segment.

One of skill in the art will understand that concentrations of the firstpeptide segment and the second and other incoming peptide segments canbe optimized using routine experimentation. Concentrations of the secondand additional incoming peptide segments can range from 1-200 mM, morepreferably from 5-100 mM, and most preferably from 10-59 mM, dependingon the particular peptide segment.

Excess first peptide segment and/or excess incoming peptide segments canbe readily removed from the solid phase bound peptide by filtration andwashing.

B. Conversion of Thioacid to Thioester using Bromoacetic Acid orIodoacetic Acid

The use of Bromoacetic acid or Iodoacetic acid is an improved method ofgenerating peptide-aCOSR thioesters from peptide-αCOSH thioacids. Inorder to insure solubility of long unprotected peptides, 6 Mguanidine-HCL at near pH 4 is used. Reactions is carried out near pH 4.Under such conditions, the only group reactive with Bromoacetic acid oriodoacetic acid is the thioacid. Benzyl bromide, a hydrophobic compound,does not dissolve completely in solution, resulting in slow andheterogeneous reactions. The advantages of using bromoacetic acid oriodoacetic acid are that both are readily soluble in 6 M guanidine-HCL(an aqueous solution) at near pH 4, both result in quick completion ofthe desired reaction, both elute in the void volume of typicalreverse-phase HPLC, and allow processing of large amounts of peptidesegments.

The resin-bound peptide thioacid is thoroughly washed in 6Mguanidine.HCl, 0.1M Na acetate, 0.15M methionine, pH 4.6 and treatedwith a 50 mM solution of bromoacetic acid in the same buffer for 15 min,followed by thorough washing with the pH 4.6 buffer (FIG. 16B, Step #3).

C. Cleavage from the Solid Phase

Cleavable handles useful in the ligations in the N- to C-terminaldirection must be capable of being stable to ligation conditions, stableto stepwise solid phase chemistries, able to be covalently linked inunprotected form to the solid phase, and be cleavable without damagingthe assembled polypeptide. Any cleavable handles satisfying theserequirements can be used, including, but not limited to: MSC handle,photolabile linkers, CAM esters (—OCHCONH—), (—O—CH2-Ø—SO—CH2-CO—),(—O—CRH—CO-Å—O-CH2-CO—). For example, (—O—CH2-Ø—SO—CH2-CO—) may be usedas a handle cleavable under any of the following conditions: (1) HF,DMS; (2) SciCl4, TFA; or red of Sulfoxide and TFA cleavage; (3) NaOH,water; or (4) red of sulfoxide and TBAF in DMF. See Samamen, J. M., J.Org. Chem. 53:561 (1988). As another example, the(—O—CRH—CO-Ø-—O—CH2-CO—) may be used as a cleavable handle under any ofthe following conditions: (1)NaOH, water(CAM Linker); (2)ZnCH3COOH/Water; (3) photolysis. See Tjoeng et al., Synthesis 897(1981); Sheehan et al., J. Org. Chem. 38:3771 (1973); Serebryakov etal., Tetrahedron 34:345 (1978); Hendrickson et al., Tetrahedron Lett.343 (1970); Ceccato, M. L. et al., Tetrahedron Lett. 31:6189-6192(1990); J. Martinez et al., Tetrahedron Lett. 41:739 (1985). One ofskill in the art will readily appreciate the suitability of knowncleavable handles for the purposes described herein.

The following conditions can be used for cleavage of the linker torelease the assembled polypeptide from the solid phase, particularlywhen an MSC handle is used. Aliquots of resin-bound peptide are treatedwith 6M guanidine.HCl, 0.1M Na acetate, 0. 15M methionine, containing200 mM hydrazine, at pH˜14 for 2 min, followed by washing with an equalamount of 6M guanidine.HCl, 0.1M Na acetate, 0.1 5M methionine, pH˜2 andan equal amount of 6M guanidine.HCl, 0.1M Na acetate, 0.15M methionine,pH 4.6. The combined eluants of free peptide are analyzed by analyticalHPLC and electrospray mass spectrometry (FIG. 16B, Step #5).

II. Solid Phase Ligations in the C- to N-Terminal Direction

The discussion regarding N- to C-terminal ligations above appliesequally well to C- to N-terminal ligations, except, as shown in FIG. 23,that: (1) the first peptide segment is bound to the solid phase via itsC-terminus, i.e. the C-terminal peptide segment of the resultingassembled polypeptide is the one modified with a cleavable handle and(2) the incoming (i.e. second, third, additional) peptide segments dorequire temporary protection of their N-terminal Cysteine (see steps2-4). Optionally, all Cysteine residues of the incoming or middlepeptide segments can be temporarily protected along with the N-terminalCysteine.

As outlined in the scheme (FIG. 23), the C-terminal peptide segmentbearing a cleavable handle is coupled to the solid support by reactionwith a corresponding functional group on the solid support (e.g. resin),for example, through an oxime linkage (aminooxyacetyl group on the resinand a ketone [via levulinic acid] on the peptide), or the reverse(aminooxyacetyl group on the peptide and a ketone on the solid phase).

Once the first peptide segment is bound to the solid phase as shown instep 1 of FIG. 21, the incoming (second) peptide segment, comprising anN-terminal protected Cys (PG-Cys) and a C-terminal thioester, reactswith the N-terminal unprotected Cys of the resin-bound first peptidesegment through the native chemical ligation reaction. After ligation iscomplete, the protecting group of the N-terminal Cys is removed (step 3of FIG. 21), and the next peptide segment is added (step 4/2 of FIG.21). Once all ligations are complete (step 5 of FIG. 21), the handleattaching the sequentially ligated peptide to the resin is cleaved,releasing the full length peptide. This C- to N-terminal technique isapplied to the total chemical synthesis of a random peptide of artificalsequence and to human secretory phospholipase A2, group 5 (“PLA2G5”), a118 amino acid enzyme, as described below.

A. Peptide Synthesis

Peptide synthesis for solid phase sequential native chemical ligation inthe C- to N-terminal direction is essentially the same as describedabove for solid phase sequential native chemical ligation in the N- toC-terminal direction.

See Example 7 below for details re stepwise solid phase peptidesynthesis of the peptide segments.

B. Preparation of the Solid Phase

Preparation of the solid phase for the C-to N-terminal direction isidentical to that described for the N- to C-terminal direction.

C. Coupling of the Modified C-Terminal Peptide Segment to Solid Phase

Conditions for coupling the modified C-terminal peptide segment to thesolid support can be identical to that outlined for coupling of themodified N-terminal peptide in the N- to C-terminal ligations asdescribed above.

D. Ligation in the C- to N-terminal Direction

Conditions for the native chemical ligation reactions in the C- toN-terminal direction can be identical to that outlined for N- toC-terminal ligations as described above, except that the N-terminalcysteine containing peptide segment is solid phase bound and theincoming thioester containing peptide segment is in solution.

E. Cysteine Protecting Groups and Removal

Any of the known protecting groups suitable for protecting theN-terminal Cys of a peptide segment can be used, provided that they arestable to ligation conditions, stable to conditions for adding thelinker, and removable from the peptide segment under conditions that arenot harmful to the solid-phase bound peptide, the linker, the resin, orthe cleavable handle, if used. The protecting groups must also be stableto stepwise solid phase peptide synthesis conditions. An example of aprotecting group is ACM (Acetamidomethyl), which provides cysteine sidechain protection (—SCH2NHCOCH3), and can be cleaved withmercury(II)acetate, or other suitable reagents. Fmoc(9Fluorenylmethylcarbamate) provides alpha amino protection, can becleaved in 20% piperidine in DMF and works well with hydrophilicpeptides. DNPE (2-(2,4-dinitriphenyl)ethyl) provides cysteine side chainprotection and cleaves in 50% piperidine in DMF.Para-nitrobenzensulfonyl provides alpha-amino protection, and is cleavedin 1 M DBU/1 M beta-mercaptoethanol in DMF. Additional cysteineprotecting groups include, but are not limited to, Sulfmoc, NSC, Dde,Boc-Cys(Acm)-OH, Fmoc-Cys-(Mob)-OH, Boc-Cys(Fm)-OH, andBoc-Cys(DNPE)-OH, wherein Acm=acetamidomethyl, Mob=methoxybenzyl,Dnpe=2-(2,4-dinitrophenyl)ethyl, Fm=9-fluorenylmethyl. See ProtectiveGroups inOrganic Synthesis, Green, T. W. and Wuts, P. G. M. eds, (2d Ed.1991), particularly p. 293-294, 318-319; R. Merrifield, J. Org. Chem.43:4808-4816 (1978); V. V. Samukov et al., Tetrahedron Lett.35:7821-7824 (1994); B. W. Bycroft et al., J. Chem. Soc. Chem. Comm.776-777 (1993); M. Royo et al., Tetrahedron Lett., 33:2391-2394 (1992);S. C. Miller, J. Am. Chem. Soc. 119:2301-2302 (1997). Certain protectinggroups can make peptide segments insoluble. For example, certainhydrophobic peptide segments may become insoluble upon addition of aprotecting group. One of ordinary skill in the art can readily ascertainthe suitability of any particular protecting group for a peptidesegment.

Removal of Fmoc as a Cys Protecting Group

One embodiment involves removal of an Fmoc protecting group from theN-terminal Cys of a solid-phase bound peptide. After ligation with apeptide with an N-terminal Fmoc-Cys, the resin bound peptide is washedwith 6 M guanidine.HCl, 0.1 M NaPi, 0.15 M methionine, pH 7, followed bywater, followed by DMF. The resin is then treated with two aliquots of20% piperidine in DMF, 5 minutes each. The resin is then washedthoroughly with DMF, followed by water, followed by 6 M guanidine.HCl,0.1 M NaPi, 0. 15 M methionine, pH 7.

Removal of ACM as a Cys Protecting Group

After ligation with a peptide with an N-terminal Cys(ACM), the resinbound peptide is washed with 6 M guanidine.HCl, 0.1 M NaPi, 0.15 Mmethionine, pH 7, followed by 3% aqueous acetic acid. The resin is thentreated with a solution of mercury(II)acetate in 3% aqueous acetic acid(15 mgs/ml) for 30 minutes, followed by washing with 3% aqueous aceticacid. The resin is then washed with 6 M guanidine.HCl, 0.1 M NaPi, 0.15M methionine, pH 7, followed by treatment with 20% beta-mercaptoethanolin 6 M guanidine.HCl, 0.1 M NaPi, 0. 15 M methionine, pH 7 for 30 min.The resin is then washed with 6 M guanidine.HCl, 0.1 M NaPi, 0. 15 Mmethionine, pH 7.

F. Cleavage from the Solid Phase

Cleavable handles are used to cleave the solid-phase bound peptide fromthe solid phase for ligations in the N- to C-terminal direction, in theC- to N-terminal direction, and in the bidirectional approach (both N-to C-terminal ligation and C- to N-terminal ligation). For solid phasesequential native chemical ligations in the C- to N-terminal direction(and for bidirectional ligations using C- to N-terminal ligation), therequirements of cleavable handle are the same as for those useful in theN- to C-terminal direction, with the additional requirement that thecleavable handle be stable under conditions used for removal of theprotecting group from the N-terminal cysteine of the solid-phase boundpeptide.

Cleavage of a Peptide-CAM Ester Linkage to the Solid Phase

Aliquots of resin-bound peptide are washed with 8M urea, 0.1 M NaPi, pH7, followed by treatment for 2 minutes with 0.25N NaOH in the same 8Murea buffer (resulting pH˜14). The resin is then washed with an equalamount of 0.25N HCl in the same 8M urea buffer (resulting pH˜2),followed by thorough washing with the 8M urea buffer. The combinedeluants of free peptide are analyzed by HPLC and electrospray massspectrometry.

III. Bidirectional Solid Phase Sequential Native Chemical Ligation

Yet another embodiment of the invention relates to bidirectional solidphase protein synthesis that incorporates aspects of both the N- toC-terminus and C- to N-terminus sequential solid phase protein synthesisapproaches. In the bidirectional approach, a peptide segment havingeither or both an N-terminal Cysteine and/or a C-terminal thioester isattached to a solid phase via a side chain of one of its amino acidresidues. See FIGS. 25A, B, C. The peptide segment can then be ligatedat either terminus to a second peptide segment, followed by ligation atthe other terminus to a third peptide segment. In this bidirectionalapproach, if the peptide segment attached to the solid phase has both aprotected N-terminal Cysteine and a C-terminal thioester, second andthird peptide segments can be added at both ends in subsequentligations. Additional peptide segments can then be added at either endof the ligated, solid phase bound peptide. The ligations in eitherdirection are accomplished using the methods described herein forligations in either the C- to N-terminal direction or the N- toC-terminal direction.

Alternatively, the firs t peptide segment attached via one of itsinternal amino acid residues to the solid phase can be used for onlyuni-directional ligations. For example, the peptide segment attached tothe solid phase can be ligated to a second peptide segment at oneterminus, followed by one or more ligations to additional peptidesegments at the same terminus of the second peptide segment. In thisembodiment, the peptide segment bound to the solid phase can be used foreither sequential solid phase native chemical ligations in the C- toN-terminal direction or for sequential solid phase native chemicalligations in the N- to C-terminal direction. In this embodiment, thepeptide segment bound to the solid phase can be bidirectionally capable(i.e. having both a protected N-terminal Cysteine and a C-terminalthioester) while being used for unidirectional sequential ligations(i.e. having either a protected N-terminal Cysteine or a C-terminalthioester).

The first peptide segment is bound to the solid phase via a side chainof one of its amino acid residues, which is bound to a cleavable handle,which is bound to the solid phase via a functional chemical moiety thatis capable of chemoselectively forming a covalent bond with acomplementary functional chemical moiety on the solid phase, asillustrated in FIG. 25.

For example, the first peptide segment can be bound to the solid phasevia the side chains of a lysine, aspartic acid or glutamic acid, inwhich case a cleavable handle based on functionalities, such asallyloxycarbonyl (alloc) or Fmoc, i.e. cleavable under orthogonalconditions, may be used to connect the peptide segment to the solidphase via the side chain of its lysine, aspartic acid or glutamic acid.As another example, an oxime bond may be formed by the first peptidesegment and the solid phase, wherein the first peptide segment compriseseither an amino-oxy or ketone chemoselective functional group and thesolid phase comprises a complementary chemoselective functional group,such as a ketone or amino-oxy, respectively.

IV. Use of Cleavable Linkers and Mass Spectrometry to Monitor LigationReactions

Various known cleavable linkers can be used to monitor the solid phasesequential ligations. These cleavable linkers are placed between thesolid phase and the first peptide segment which is covalently bound tothe cleavable handle, e.g. solid phase—cleavable linker—cleavablehandle—peptide segment. The cleavable linkers are capable of beingreadily cleaved to permit mass spectrometric analysis of a small portionof solid phase-bound peptide to monitor the coupling and ligationreactions.

For example, when the solid phase consists of resin beads, one can takea few resin beads from the reaction mixture after the coupling reactionor after each ligation reaction to determine the extent of reaction.Particularly preferred cleavable linkers include photolabile cleavablelinkers for MALDI mass spectrometry, including3-nitro-4(methylamino)benzoyl-. See FIG. 5A. A small aliquot of thereaction mixture is removed for MALDI MS analysis and dried on a slidein mixture with a matrix solution. The laser of the MALDI massspectrometer cleaves the photolabile linker on the mass spectrometer'sstage, permitting mass analysis of the released peptides.

Another preferred cleavable linker is one that is cleavable by TFA(trifluoroacetic acid), which is useful for electrospray ionization massspectrometry. With TFA-cleavable linkers, the peptides are cleaved fromthe solid phase prior to ESI MS.

EXAMPLES Example 1 Preparation of the Solid Phase for N- to C-TerminalLigations

The preparation of the solid phase is schematically diagrammed in FIG.5. The solid phase is a resin, for example, Amino PEGA (0.2-0.4 mmol/gswelled in methanol) or an amino-Spherilose affinity resin (15-20Tmol/ml, 0.6-0.9 mmol/g dry resin), available from Pharmacia, NovaSyn orIsco. The resin (PEGA or Isco) is washed with DMF (dimethylformamide),then is washed briefly with 10% DIEA (diisopropyl ethylamine). Two 30sec DMF flow washes are used. A photocleavable linker (PCL) (See FIG.5A) is activated with one equivalent of HBTU(2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl-uroniumhexafluorophosphate) and DIEA in DMF for 5-10 min). This activatedphotocleavable linker is then added to the resin and is left standing atroom temperature for ˜3 hrs (ninhydrin can be used with Isco). Two 30sec. DMF flow washes are used, followed by TFA (1 min×2), and two more30 sec. DMF flow washes. The remaining steps are in abbreviated form:

10% DIEA (1 min×2)

DMF flow wash (30 sec×2)

addition of activated Boc-aminooxyacetic acid (activated with oneequivalent DIC and N-hydroxysuccinimide in DMF for 30-60 min)

left standing at room temperature for ˜1 hr (ninhydrin can be used withIsco)

DMF flow wash (30 sec×2) [resin can be stored at this stage]

TFA (1 min×30)

DMF flow wash (30 sec×2)

10% DIEA (1 in×2)

DMF flow wash (30 sec×2)

thorough washing with aqueous buffer (6 M GuHCl, 0.1 M Na Acetate, pH4.6) (1 ml×5)

Example 2 Preparation of the First Unprotected Peptide Segment for N- toC-terminal Ligations

The following procedures are used to prepare the first peptide segment(N-terminus), which is diagrammed in FIGS. 6, 7A and 7B.

The peptide-resin is swelled in DMF

TFA (1 min×2)

DMF flow wash (30 sec×2)

10% DIEA (1 min×2)

DMF flow wash (30 sec×2)

Addition of MSC handle in DMF

leave standing at room temperature for 1 hr

add DIEA and leave standing for another hr

use ninhydrin test to verify adequate coupling

DMF flow wash (30 sec×2)

TFA (1 min×2)

DMF flow wash (30 sec×2)

10% DIEA (1 min×2)

DMF flow wash (30 sec×2)

addition of activated levulinic acid (activated as the symmetricanhydride with 0.5 equivalents of DIC in DCM for 5-10 min)

leave standing at room temperature for 30 min

ninhydrin test to verify adequate ligating

DMF flow wash (30 sec×2)

thorough washing with DCM

dry on lyophilizer

HF cleavage at 0° C. for 1 hr using p-cresol as a scavenger

trituration and washing with cold ethyl acetate

dissolve in 50% B and lyophilize

purify by preparative HPLC

TABLE 1 Solid Phase Sequential Ligations: N- to C-Terminal 3-RandomPeptide Segment Model SystemLev-MSC-LTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSRKHG-COS) (1) + Resin-PCL-ONH₂↓1. pH 4.6, 6 M GuHCl, 0.1 M acetateResin-PCL-oxime-MSC-LTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSRKHG-COS) (1) ↓2. pH4.6, 6 M GuHCl, 0.1 M acetate, 50 mM BrAcOHResin-PCL-oxime-MSC-LTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSRKHG-COSAc (1) +H-CGFRVREFGDNTA-COS) (2) ↓3. pH 7.5, 6 M GuHCl; 0.1 M phosphate, 0.5%thiophenol Resin-PCL-oxime-MSC-LTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSRKHGCGFRVREF-GDNTA-COS) (1 + 2) ↓4. pH 4.6, 6 M GuHCl, 0.1 M acetate, 50 mMBrAcOH Resin-PCL-oxime-MSC-LTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSRKHGCGFRVREF-GDNTA-COSAc (1 + 2) + H-CADPSEEWVQKYVSDLELSA-OH (3) ↓5. pH 7.5,6 M GuHCl, 0.1 M phosphate, 0.5% thiophenolResin-PCL-oxime-MSC-LTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSRKHGCGFRVREF-GDNTACADPSEEWVQKYVSDLELSA-OH (1 + 2 + 3) ↓6. pH 14, 6 M GuHCl,0.1 M phosphate, 200 mM hydrazineH-LTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSRKHGCGFRVREF-GDNTACADPSEEWVQKYVSDLELSA-OH (l + 2 + 3) (SEQ ID NO:2) PCL =photocleavable linker

Example 3 Solid Phase Native Chemical Ligation of Random PeptideSegments in Aqueous Solution in the N- to C-terminus Direction

The following procedures are used for solid phase ligations in the N- toC-terminus direction, as diagrammed in Table 1. General principals ofnative chemical ligation are described in WO 96/34878, PCT/US95/05668,incorporated herein by reference.

The resin is washed with 6 M guanidine.HCl, 0.1 M Na Acetate, pH 4.6 (1ml×5) and drained. The modified N-terminal peptide segment is dissolvedin 6 M guanidine.HCl, 0.1 M Na Acetate, pH 4.6 and added to resin and isleft standing at room temperature overnight. (The concentration of thefirst segment is at least 5 mM). The next morning, resin is washed with6 M guanidine.HCl, 0.1 M Na Acetate, pH 4.6 (1 ml×5) and drained. Asample of resin is removed for MALDI MS analysis and is washed with50%B, MeOH, DCM and dried. A sample of resin is removed for basecleavage and is treated with 200 μl 6 M guanidine.HCl, 0.1 M Na Pi, 200mM hydrazine, pH˜14 for 2 min and drained, resin is washed with 200 μl 6M guanidine.HCl, 0.1 M Na acetate, 200 mM hydrazine, pH˜2 and with 200μl 6 M guanidine.HCl, 0.1 M Na Acetate, pH 4.6 and the combined eluantstreated with TCEP prior to injection on HPLC.

In preparation for addition of the next peptide segment, the resin iswashed with 6 M guanidine.HCl, 0.1 M Na Pi, pH 7.5 (1 ml×5) and drained.The second peptide segment (Cys-COSH) is dissolved in 6 M guanidine.HCl,0.1 M Na Pi, pH 7.5, 0.5% thiophenol and added to resin. This mixture isleft standing at room temperature overnight. The next morning, the resinis washed with 6 M guanidine.HCl, 0.1 M Na Acetate, pH 4.6 (1 ml×5) anddrained. Samples of resin are removed for Maldi and base cleavage andtreated as above

The solid phase-bound peptide is then converted from COSH to COSAc bytreating the resin with 50 mM BrAcOH in 6 M guanidine.HCl, 0.1 M NaAcetate, pH 4.6 for 15 min.

The resin is washed with 6 M guanidine.HCl, 0.1 M Na Acetate, pH 4.6 (1ml×5) and drained.

In preparation for addition of the next peptide segment, the resin iswashed with 6 M guanidine.HCl, 0.1 M Na Pi, pH 7.5 (1 ml×5) and drained.The final peptide segment is dissolved in 6 M guanidine.HCl, 0.1 M NaPi, pH 7.5, 0.5% thiophenol and added to resin. This reaction mixture isleft standing at room temperature overnight. The next morning, the resinis washed with 6 M guanidine.HCl, 0.1 M Na Acetate, pH 4.6 (1 ml×5) anddrained. A sample of resin are removed for monitoring by MALDI MSanalysis.

The assembled peptide is removed from the solid phase via base cleavageof the cleavable handle from the remaining resin as outlined above onlyon a larger scale followed by purification by HPLC or desalting on PD-10column and lyophilization.

Example 4 Solid Phase Native Chemical Ligation of C5a(1-74) (74aa) inthe N- to C-Terminal Direction

This example describes solid phase sequential native chemical ligationin the N- to C-terminal direction of C5a, Complement Factor 5A. Thesequence of C5a is:

TLQKKIEEIAAKYKJSVVKKCCYDGACVNNDETCEQRAARISLGPKCIKAFTECCVVASQLRANISHKDMQLGR(SEQ ID NO:3).

This peptide is prepared using solid phase sequential native ligation of3 peptide segments: C5a(1-20), C5a(21-46), and C5a(47-74). Theprocedures used to synthesize C5a by solid phase ligations are identicalto those described in the solid phase sequential native ligation of MIF(See Example 5).

Example 5 Solid Phase Sequential Native Chemical Ligation of MIF(1-115)(115 aa) in the N-Terminal to C-Terminal Direction

The sequence of MIF(1-115) is:MPMFIVNTNVPRASVPDGFLSELTQQLAQATGKPPQYIAVHVVPDQLMAFGGSSEPCALCSLHSIGKIGGAQNRSYSKLLCGLLAERLRISPDRVYINYYDMNAASVGWNNSTFA(SEQ ID NO:4). This peptide is prepared using solid phase sequentialnative ligation of 3 peptide segments: MIF(1-59) (amino acids 1-59; SEQID NO:4), MIF(60-80) (amino acids 60-80; SEQ ID NO:4) and MIF(81-115)(amino acids 81-115; SEQ ID NO:4). See FIGS. 16-20.

Step #1: The first unprotected peptide segment, MIF(1-59) is coupled toa solid phase as depicted in FIG. 18. The coupling conditions are 6Mguanidine.HCl, 0.1M NaAcetate, 0.15M Methionine, pH 4.6, 24 hours.

The MSC handle used is:

This cleavable handle is based on methylsulfonylethyloxycarbonyl (MSC)amine protecting group. It is easily added to unprotected amino terminusof peptide-resins, survives HF deprotection and cleavage from the resin,is quickly and cleanly cleaved by aqueous base, and is designed with aprotected amine which can be derivatized with a variety offunctionalities.

Step #2: The second unprotected peptide segment (Cys60-MIF(61-80)-COSH)is then ligated to the solid phase-bound first unprotected peptidesegment, under the conditions 6 M guanidine.HCl, 0.1M NaPi, 0.5%thiophenol, 0.1 SM Methionine, pH 7.5, 24 hours.

Step #3: The solid phase-bound peptide, MIF(1-80)-COSH, is thenactivated to the thioester under the following conditions: 50 mMBrCH2COOH, 6M guanidine.HCl, 0.1M NaAcetate, 0.15M Methionine, pH 4.6,15 min.

Step #4: The third unprotected peptide segment (Cys81-MIF82-115-COOH) isligated to the solid phase-bound peptide with 6 M guanidine.HCl, 0.1MNaPi, 0.5% thiophenol, 0.15M Methionine, pH 7.5, 24 hours.

Step #5: The MIF(1-115) bound to the solid phase is then cleaved fromthe solid support by base cleavage of the cleavable handle under thecleaving conditions: 6 M guanidine.HCl, 0.1M NaAcetate, 0.1 5MMethionine, 200 mM hydrazine, at pH˜14 for 2 min., followed by 6 Mguanidine.HCl, 0.1M NaAcetate, 0.15M Methionine, 200 mM hydrazine, atpH˜2. The expected mass of the assembled peptide MIF(1-115) releasedupon base cleavage is 12450 Da. FIGS. 20C and 20D are mass spectra ofthe assembled peptide having an expected mass of 12450. FIG. 20D is areconstruction of the mass spectrum of FIG. 20C. FIG. 20B is an HPLCchromatogram of the assembled peptide.

Example 6 Solid Phase Native Chemical Ligation of Phospholipase A2,Group 5(1-118) (118aa) in the C- to N-terminal Direction

The sequence of Phospholipase A2, group 5 (PLA2G5) is:GLLDLKSMIEKVTGKNALTNYGFYGCYCGWGGRGTPKDGTDWCCWAHDHCYGRLEEKGCNIRTQSYKYRFAWGVVTCEPGPFCHVNLCACDRKLVYCLKRNLRSYNPQYQYFPNILCS(SEQ ID NO:5).

This peptide is prepared using solid phase sequential native ligation of4 peptide segments: PLA2G5 (1-25), PLA2G5 (26-58), PLA2G5(59-87) andPLA2G5 (88-118). The procedures used to synthesize PLA2G5 by solid phaseligations are identical to those used for synthesizing the randomsequence using ACM protection of the N-terminal Cys residues of themiddle segments, as described in Example 9. See FIG. 22 for the reactionscheme. The Cam ester derivative is synthesized and incorporated intothe C-terminal peptide segment according to the diagrams in FIGS. 23,24/FIG. 27. The assembled polypeptide, PLA2G5 (1-118), was folded andassayed for biological activity. It had the full activity of arecombinantly expressed PLA2G5.

Example 7 Preparation of Modified C-terminal Peptide Segment (On-resinCAM Linker Synthesis) (FIG. 27)

The commercial resin of choice (MBHA, any Boc-AA-OCH2-Pam resin) isswelled in DMF

TFA (1 min×2) (not necessary if working with MBHA resin)

DMF flow wash (30 sec×2)

addition of activated Boc-Lys(Fmoc)-OH (HBTU/DIEA activation), check forcompletion of reaction after 10-15 minutes by ninhydrin test

DMF flow wash (30 sec×2)

TFA (1 min×2)

DMF flow wash (30 sec×2)

10% DIEA in DMF (1 min×2)

addition of activated bromoacetic acid (activated as the symmetricanhydride with 0.5 equivalents of DIC in DCM for 5-10 minutes), checkfor completion of reaction after 30 minutes by ninhydrin test

DMF flow wash (30 sec×2)

addition of first Boc-protected amino acid of the sequence(Boc-AA-OH) 2Min 20% DIEA in DMF. Leave standing at room temperature for 3 hrs.

DMF flow wash (30 sec×2)

synthesize rest of the sequence by standard protocols for Boc chemistry

remove Fmoc group by treating with 20% piperidine in DMF (5 min×2)

DMF flow wash (30 sec×2)

addition of activated levulinic acid (activated as the symmetricanhydride with 0.5 equivalents of DIC in DCM for 5-10 min), check forcompletion of reaction after 30 minutes by ninhydrin test

DMF flow wash (30 sec×2)

thorough washing with DCM

thoroughly dry resin

HF cleavage at 0° C. for 1 hr using p-cresol as a scavenger

trituration and washing with cold ethyl ether

dissolve in aqueous HPLC buffer and lyophilize

purify by preparative HPLC

Example 8 Solid Phase Native Chemical Ligation of Random PeptideSegments in the C- to N-terminal Direction using Fmoc Protection (SeeFIG. 28)

The following procedures can be used for solid phase ligations in the C-to N-terminal direction, as diagramed in Table 2. By example, a randompeptide of: ALTKYGFYGCYGRLEEKGCADRKNILA (SEQ ID NO:1) can be ligated inthree peptide segments (from C- to N-terminal direction): segment1=CADRKNILA (amino acids 19-27; SEQ ID NO:1); segment 2=CYGRLEEKG (aminoacids 10-18; SEQ ID NO:1); and segment 3=ALTKYGFYG (amino acids 1-9; SEQID NO:1).

The resin is washed with 6M Gu.HCL, 0.1M Na Acetate, pH 4.6 (1 ml×5) anddrained. The modified C-terminal peptide segment (first peptide segment)is dissolved in 6M Gu.HCL, 0.1M Na Acetate, pH 4.6 (5 mM first peptidesegment) and added to the resin and is left standing at room temperatureovernight. The resin is washed with 6M Gu.HCL, 0.1M Na Acetate, pH 4.6(1 ml×5) and drained. A sample is removed for base cleavage and istreated with 8M urea, 0.1M NaPi, pH 7, treated for 2 minutes with 0.25NNaOH in the same 8M urea buffer (resulting pH˜14), washed with an equalamount of 0.25N HCl in the same 8M urea buffer (resulting pH˜2), and thecombined eluants treated with TCEP prior to injection on HPLC.

In preparation for addition of the next segment, the resin is washedwith 6M Gu-HCl, 0.1M NaPi, pH 7.0 (1 ml×5) and drained. The secondpeptide segment (Fmoc-Cys-peptide-COSR) is dissolved in 6M Gu.HCl, 0.1MNaPi, pH 7.0, 0.5% thiophenol (to at least 10 mM to 50 mM second peptidesegment) and added to the resin. The mixture is left standing at roomtemperature overnight. The resin is washed with 6M Gu.HCl, 0.1M NaPi, pH7.0 (1 ml×5), water (1 ml×5), DMF (1 ml×5), and the Fmoc protectinggroup removed by treating with two aliquots of 20% piperidine in DMF (5min each). The resin is then washed with DMF (1 ml×5), water (1 ml×5),and 6M Gu.HCl, 0.1M NaPi, pH 7.0 (1 ml×5). A sample of resin is removedand base cleaved as above.

The final peptide segment is dissolved in 6M Gu.HCl, 0.1M NaPi, pH 7.0,0.5% thiophenol and added to the resin. This mixture is left standing atroom temperature overnight. The resin is then washed with 6M Gu.HCl,0.1M NaPi, pH 7.0 and the assembled peptide is removed from the solidphase via base cleavage of the cleavable handle from the remaining resinas outlined above only on a larger scale followed by purification byHPLC or deslating on PD-10 column and lyophilization.

These methods can be applied to make any peptides having cysteineresidues.

Example 8A Solid Phase Native Chemical Ligation of Random PeptideSegments in the C- to N-terminal Direction using DNPE Protection

DNPE (2-(2,4-dinitrophenylethyl)) is another cysteine side chainprotecting group which can be used for ligations in the C- to N-terminaldirection. Example 8 was repeated using DNPE as the protecting group.The conditions for solid phase chemical ligation of random peptidesegments in the C- to N-terminal direction were identical to those usedfor Example 8 above except that in the removal of the DNPE protectinggroup, 50% piperidine is used.

Example 9 Solid Phase Native Chemical Ligation of Random PeptideSegments in the C- to N-terminal Direction using ACM Protection

The following procedures are used for solid phase ligations in the C- toN-terminal direction, as diagramed in Table 3. The same randompolypeptide described in the Example above is ligated.

The resin is washed with 6M Gu.HCL, 0.1M Na Acetate, pH 4.6 (1 ml×5) anddrained. The modified C-terminal peptide segment is dissolved in 6MGu.HCL, 0.1M Na Acetate, pH 4.6 and added to the resin and is leftstanding at room temperature overnight. The resin is washed with 6MGu.HCL, 0.1M Na Acetate, pH 4.6 (1 ml×5) and drained. A sample isremoved for base cleavage and is treated with 8M urea, 0.1M NaPi, pH 7,treated for 2 minutes with 0.25N NaOH in the same 8M urea buffer(resulting pH˜14), washed with an equal amount of 0.25N HCl in the same8M urea buffer (resulting pH˜2), and the combined eluants treated withTCEP prior to injection on HPLC

In preparation for addition of the next segment, the resin is washedwith 6M Gu.HCl, 0.1M NaPi, pH 7.0 (1 ml×5) and drained. The secondpeptide segment (Fmoc-Cys-peptide-COSR) is dissolved in 6M GuHCl, 0.1MNaPi, pH 7.0, 0.5% thiophenol (to at least 10 mM second peptide segment)and added to the resin. The mixture is left standing at room temperatureovernight. The resin is washed with 6M Gu.HCl, 0.1M NaPi, pH 7.0 (1ml×5), 3% acetic acid in water (1 ml×5), and the ACM protecting groupremoved by treating with mercury(II)acetate in 3% acetic acid in water(15 mgs/ml) for 30 min. The resin is then washed with 3% acetic acid inwater (1 ml×5), 6M Gu.HCl, 0.1M NaPi, pH 7.0 (1 ml×5), and treated with20% beta-mercaptoethanol in 6M Gu.HCl, 0.1M NaPi, pH 7.0 for 30 min,followed by washing with 6M Gu.HCl, 0.1M NaPi, pH 7.0 (1 ml×5). A sampleof resin is removed and base cleaved as above. The final peptide segmentis dissolved in 6M Gu.HCl, 0.1M NaPi, pH 7.0, 0.5% thiophenol and addedto the resin. This mixture is left standing at room temperatureovernight. The resin is then washed with 6M Gu.HCl, 0.1M NaPi, pH 7.0and the assembled peptide is removed from the solid phase via basecleavage of the cleavable handle from the remaining resin as outlinedabove only on a larger scale followed by purification by HPLC ordeslating on PD-10 column and lyophilization.

TABLE 2 Polymer-Supported Ligations C- to N- Terminal Direction FmocProtection H-CADRKNILA-CAM-Lys(Levulinic acid)-NH₂ ₍₁₎ ₊ Resin- ONH₂ ↓1.pH 4.6, 6 M Gu.HCl, 0.1 acetate H-CADRKNILA-CAM-Lys-oxime-Resin (1) +Fmoc-CYGRLEEKG-COSR (2) ↓2. pH 7.5, 6 M Gu.HCl, 0.1 M phosphate, 0.5%thiophenol Fmoc-CYGRLEEKGCADRKNILA-CAM-Lys-oxime-Resin (1 + 2) ↓3. 20%piperidine/DMF H-CYGRLEEKGCADRKNILA-CAM-Lys-oxime-Resin (1 + 2) +H-ALTKYGFYG-COSR (3) ↓4. pH 7.5, 6 M Gu.HCl, 0.1 M phosphate, 0.5%thiophenol H-ALTKYGFYGCYGRLEEKGCADRKNILA-CAM-Lys-oxime-Resin (1 + 2 + 3)↓5. pH 14, 8 M Urea, 0.1 M phosphate, 0.25 N NaOHH-ALTKYGFYGCYGRLEEKGCADRKNILA-OH(SEQ ID NO:1)

TABLE 3 Polymer-Supported Ligations C- to N- Terminal Direction ACMProtection H-CADRKNILA-CAM-Lys(Levulinic acid)-NH2 (1) + Resin-ONH₂ ↓1.pH 4.6, 6 M Gu.HCl, 0.1 acetate H-LCADRKNILA-CAM-Lys-oxime-Resin (1) +H-C(ACM)YGRLEEKG-COSR (2) ↓2. pH 7.5, 6 M Gu.HCl, 0.1 M phosphate, 0.5%thiophenol H-C(ACM)YGRLEEKGCADRKNILA-CAM-Lys-oxime-Resin (1 + 2) ↓3. a.mercury(II)acetate in 3% Aq. AcOH b. 20% mercaptoethanol in pH 7.5, 6 MGu.HCl, 0.1 M phosphate H-CYGRLEEKGCADRKNILA-CAM-Lys-oxime-Resin (1 +2) + H-ALTKYGFYG-COSR (3) ↓4. pH 7.5, 6 M Gu.HCl, 0.1 M phosphate, 0.5%thiophenol H-ALTKYGFYGCYGRLEEKGCADRKNILA-CAM-Lys-oxime-Resin (1 + 2 + 3)↓5. pH 14, 8 M Urea, 0.1 M phosphate, 0.25 N NaOHH-ALTKYGFYGCYGRLEEKGCADRKNILA-OH(SEQ ID NO:1)

Example 10 Bidirectional Solid Phase Sequential Native Chemical Ligation

This example illustrates one of the embodiments of the bidirectionalsolid phase protein ligation approach, namely the situation startingwith a first peptide segment bound to the solid phase, wherein the firstpeptide segment is a “middle piece” of the target protein desired, i.e.The first peptide segment, bound to the solid phase, is used forligations at both its N-terminal Cysteine and its C-terminal thioester.

Starting with one of the middle pieces of the target protein, acleavable linker is added to the side chain of one of the amino acidresidues of the middle piece. The side chain of any amino acid residuehaving a protectable functional group can be used, including, preferablyAspartic Acid or Glutamic Acid. Most preferably, a Lysine amino acidresidue is used. For example, a CAM ester cleavable handle or any othercarboxylic acid protecting group may be adapted to attach the firstpeptide segment to the solid phase through the side chain of Aspartic orGlutamic Acid. One of skill in the art will readily appreciate thenecessary chemistries for accomplishing this step.

For example, the synthesis of a first peptide segment to be attached tothe solid phase via an internal amino acid is illustrated in FIG. 25C.Starting with an appropriate solid phase (thioester or thiacidgenerating), the first peptide segment is synthesized using standard Bocprotocols until the Lysine residue of choice is reached. Using Bocchemistry, a Lysine with its side chain amine protected with an Fmocgroup (Boc-Lys(Fmoc)-OH) is inserted at the appropriate location duringsolid phase stepwise peptide synthesis, followed by continued synthesisto the end of the first peptide segment. The Fmoc protecting group isremoved at the end of the stepwise peptide synthesis and the cleavablehandle coupled to the side chain amine (step B of FIG. 25C).

This method is much the same as the procedure outlined in FIG. 24, withthe following differences: the levulinic acid in step 4 is replaced withthe cleavable handle and the 20% piperidine used to cleave the Fmocgroup (also part of step 4) is replaced with a much smallerconcentration of an alternative base such as1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU), e.g. 1-2 equivalents of DBU inDMF. The reason is the middle peptide segments, regardless of whetherthey generate thioacids or thioesters upon cleavage from the resin, areconnected to the resin by a thioester which would be cleaved in thepresence of 20% piperidine.

For this particular strategy, the MSC handle is preferred, althoughother cleavable handles can be used. Attachment to the side chain amineof a lysine residue and further modification of the linker with anappropriate functional group capable of reacting with a correspondinggroup on the solid phase ligation resin would be generally as outlinedin FIG. 17A, with the exception that the amine of the MSC handle shouldbe protected with an Fmoc instead of a Boc group. Since attachment tothe peptide segment is through an internal amino acid residue, theN-terminal amino acid would be Boc protected and it is not possible forthe N-terminal amino group and the amino group of the MSC cleavablehandle to be protected by the same group. Removal of the Fmoc group onthe MSC cleavable handle would also need to be done with DBU instead ofpiperidine. As in FIG. 17A, levulinic acid is preferred for coupling tothe linker with a corresponding aminooxyacetyl group on the solidsupport (FIG. 17B).

Two versions of the first peptide segment to be coupled to the resin aredescribed below.

First Version

The first peptide segment has an unprotected N-terminal cysteine and aC-terminal thioacid (FIG. 25A). The second peptide segment (step 2 inFIG. 25A), to be ligated to the first peptide segment, is a peptide witha C-terminal thioester and optionally a protected N-terminal Cysteine(if additional C- to N-terminal ligations are desired), wherein theC-terminal thioester is capable of reacting with the N-terminal Cys ofthe first peptide segment (i.e. in the C- to N-terminal direction). Thisstep can be multiply repeated with additional peptide segments added inthe C- to N-terminal direction, if desired, provided that the internalincoming peptide segments each comprise a protected N-terminal Cysteine,which can be deprotected according to the standard C- to N-terminalsolid phase native chemical ligation steps outlined in FIG. 21 (thefinal peptide segment to be added at the N-terminus of the resultingproduct need not have an N-terminal Cysteine). After ligation iscomplete, the C-terminal thioacid of the resulting solid-phase boundpeptide (i.e. ligation product of first and second peptide segments) isthen converted to a thioester with bromoacetic acid (as outlined in N-to C-terminal ligations in Table 1 and diagrammed as step 3 of FIG.25A). The next step (step 4 of FIG. 25A) comprises ligation of thesolid-phase bound peptide to a third peptide segment with an N-terminalCys. This step can optionally be repeated, to add additional incomingpeptide segments in the N- to C-terminal direction, if desired, providedthat the internal incoming peptide segments each comprise an unprotectedN-terminal Cysteine and a C-terminal thioacid, with conversion of thethioacid to thioester after the ligation is complete and prior toaddition of the next peptide segment. The final peptide segment to beadded at the C-terminus of the resulting product need not have aC-terminal thioacid.

One of skill in the art will appreciate that multiple ligations cansubsequently be performed in both directions if the appropriateprotecting groups and other appropriate chemistries are used on themiddle piece or the solid-phase bound peptide. These additional stepsare identical to the strategies used for the individual directions, i.e.N-terminal unprotected Cys plus C-terminal thioester for the N- to C-direction and N-terminal Cys(ACM) plus C-terminal thioester for the C-to N-terminal direction. Assuming the MSC linker is used, cleavage ofthe full length product from the resin would be in basic solution (pH12-14) as outlined in step 6 in Table 1. However, the preferred approachis to complete all ligation steps necessary for one direction, followedby the ligation steps for the other direction. As long as the solidphase bound peptide has either a protected N-terminal Cysteine or aC-terminal thioacid, ligations can proceed in either direction providedthat the appropriate strategies as described herein are followed. If thesolid phase bound peptide has both an unprotected N-terminal Cysteineand a C-terminal thioester, any attempts at ligating to an additionalincoming peptide segment will result in cyclization of the solid-phasebound peptide.

Second Version

The second version of this scheme involves starting with ligation in theN- to C-terminal direction, followed by ligation in the oppositedirection, as shown in FIG. 25B. The first peptide segment to be coupledto the resin comprises a temporarily protected N-terminal Cys and aC-terminal thioester. The ligation of a second peptide segment to thefirst peptide segment is then in the N- to C-terminal direction. Anysubsequent ligations in the C- to N-terminal direction would firstrequire removal of the protecting group.

Except for the attachment of the first peptide segment to the solidsupport, this strategy merely combines the procedures for N- to C- andC- to N-terminal ligations (described above).

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PCT/US94/07222, WO 95/00846, Published Jan. 5, 1995.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the appendedclaims.

6 1 27 PRT Artificial Sequence Description of ArtificialSequencesynthetic 1 Ala Leu Thr Lys Tyr Gly Phe Tyr Gly Cys Tyr Gly ArgLeu Glu Glu 1 5 10 15 Lys Gly Cys Ala Asp Arg Lys Asn Ile Leu Ala 20 252 68 PRT Artificial Sequence Description of Artificial Sequencesynthetic2 Leu Thr Glu Gly Leu His Gly Phe His Val His Glu Phe Gly Asp Asn 1 5 1015 Thr Ala Gly Cys Thr Ser Ala Gly Pro His Phe Asn Pro Leu Ser Arg 20 2530 Lys His Gly Cys Gly Phe Arg Val Arg Glu Phe Gly Asp Asn Thr Ala 35 4045 Cys Ala Asp Pro Ser Glu Glu Trp Val Gln Lys Tyr Val Ser Asp Leu 50 5560 Glu Leu Ser Ala 65 3 73 PRT Homo sapiens 3 Thr Leu Gln Lys Lys IleGlu Glu Ile Ala Ala Lys Tyr Lys Ser Val 1 5 10 15 Val Lys Lys Cys CysTyr Asp Gly Ala Cys Val Asn Asn Asp Glu Thr 20 25 30 Cys Glu Gln Arg AlaAla Arg Ile Ser Leu Gly Pro Lys Cys Ile Lys 35 40 45 Ala Phe Thr Glu CysCys Val Val Ala Ser Gln Leu Arg Ala Asn Ile 50 55 60 Ser His Lys Asp MetGln Leu Gly Arg 65 70 4 115 PRT Homo sapiens 4 Met Pro Met Phe Ile ValAsn Thr Asn Val Pro Arg Ala Ser Val Pro 1 5 10 15 Asp Gly Phe Leu SerGlu Leu Thr Gln Gln Leu Ala Gln Ala Thr Gly 20 25 30 Lys Pro Pro Gln TyrIle Ala Val His Val Val Pro Asp Gln Leu Met 35 40 45 Ala Phe Gly Gly SerSer Glu Pro Cys Ala Leu Cys Ser Leu His Ser 50 55 60 Ile Gly Lys Ile GlyGly Ala Gln Asn Arg Ser Tyr Ser Lys Leu Leu 65 70 75 80 Cys Gly Leu LeuAla Glu Arg Leu Arg Ile Ser Pro Asp Arg Val Tyr 85 90 95 Ile Asn Tyr TyrAsp Met Asn Ala Ala Ser Val Gly Trp Asn Asn Ser 100 105 110 Thr Phe Ala115 5 118 PRT Homo sapiens 5 Gly Leu Leu Asp Leu Lys Ser Met Ile Glu LysVal Thr Gly Lys Asn 1 5 10 15 Ala Leu Thr Asn Tyr Gly Phe Tyr Gly CysTyr Cys Gly Trp Gly Gly 20 25 30 Arg Gly Thr Pro Lys Asp Gly Thr Asp TrpCys Cys Trp Ala His Asp 35 40 45 His Cys Tyr Gly Arg Leu Glu Glu Lys GlyCys Asn Ile Arg Thr Gln 50 55 60 Ser Tyr Lys Tyr Arg Phe Ala Trp Gly ValVal Thr Cys Glu Pro Gly 65 70 75 80 Pro Phe Cys His Val Asn Leu Cys AlaCys Asp Arg Lys Leu Val Tyr 85 90 95 Cys Leu Lys Arg Asn Leu Arg Ser TyrAsn Pro Gln Tyr Gln Tyr Phe 100 105 110 Pro Asn Ile Leu Cys Ser 115 6 10PRT Artificial Sequence Description of Artificial Sequencesynthetic 6Asp Ser Val Ile Ser Leu Ser Gly Asp His 1 5 10

What is claimed is:
 1. A method of producing an assembled peptide in aqueous solution and on a solid phase comprising: a) binding a partially or completely unprotected first peptide segment to a solid phase via a linker to form a solid phase-bound first peptide segment, wherein said partially or completely unprotected first peptide segment comprises an N-terminus and a thioester of the formula —COSR at its C-terminus, wherein said linker comprises a cleavable moiety and said partially or completely unprotected first peptide segment is bound to said linker at said N-terminus, and wherein R is a straight or branched C₁₋₁₅ functionalized alkyl group, a C₁₋₁₅ aromatic structure, or 1 to 4 amino acids or derivatives thereof; b) ligating a partially or completely unprotected second peptide segment to said solid phase-bound first peptide segment, wherein said second peptide segment comprises a cysteine at its N-terminus and a thioacid at its C-terminus, and wherein said N-terminal cysteine of said second peptide segment is capable of selectively ligating to said C-terminus of said solid phase-bound first peptide to form a solid phase-bound peptide comprising a thioacid at its C-terminus; c) converting said thioacid of said phase-bound peptide to a thioester of the formula —COSR to form a solid phase-bound peptide comprising a thioester at its C-terminus; and d) ligating an partially or completely unprotected third peptide segment to said solid phase-bound peptide, wherein said third peptide segment comprises a cysteine at its N-terminus, to form a solid phase-bound assembled peptide; and e) optionally repeating steps to b), c) and d) with additional partially or completely unprotected peptide segments, wherein if said steps b) and c) are repeated, the peptide segment of step d) prior to such repetition comprises a thioacid at its C-terminus.
 2. The method of claim 1, further comprising, after step d), cleaving said linker to release said assembled peptide.
 3. The method of claim 1, wherein said assembled peptide is from 20 to 1000 amino acids in length.
 4. The method of claim 1, wherein said solid phase is a bead resin.
 5. The method of claim 1, wherein said cleavable moiety is a cleavable handle.
 6. The method of claim 1, wherein said cleavable moiety is a cleavable linker.
 7. The method of claim 1, wherein said first, second and third peptide segments range in size from 5 to 99 amino acids residues.
 8. The method of claim 1, wherein said first, second and third peptide segments are all prepared by stepwise solid phase synthesis.
 9. The method of claim 1, wherein the last peptide segment to be ligated onto the solid phase-bound peptide is derived from recombinant DNA expression.
 10. The method of claim 1, wherein at least one of said first, second and third peptide segments comprises an unnatural backbone structure.
 11. The method of claim 1, wherein said converting step is accomplished using bromoacetic acid.
 12. The method of claim 1, further comprising monitoring one or more of the ligation reactions of said ligating using mass spectrometric analysis.
 13. A method of producing an assembled peptide comprising: a) binding a partially or completely unprotected first peptide segment to a solid phase via a linker, wherein said partially or completely unprotected first peptide segment comprises an N-terminus and a thioacid of the formula —COSH at its C-terminus, wherein said linker comprises a cleavable moiety and said partially or completely unprotected first peptide segment is bound to said linker at said N-terminus; b) converting said C-terminal thioacid of said first peptide segment to a thioester of the formula —COSR, wherein R is a straight or branched C₁₋₁₅ functionalized alkyl group, a C₁₋₁₅ aromatic structure, or 1 to 4 amino acids or derivatives thereof; c) ligating a partially or completely unprotected second peptide segment to said solid phase-bound first peptide segment, wherein said second peptide segment comprises a cysteine at its N-terminus and a thioacid at its C-terminus, and wherein said N-terminal cysteine of said second peptide segment is capable of selectively ligating to said C-terminus of said solid phase-bound first peptide segment to form a solid phase-bound peptide comprising a thioacid at its C-terminus; d) converting said thioacid of said phase-bound peptide to a thioester of the formula —COSR to form a solid phase-bound peptide comprising a thioester at its C-terminus; e) ligating a partially or completely unprotected third peptide segment to said solid phase-bound peptide, wherein said third peptide segment comprises a cysteine at its N-terminus, to form said assembled peptide; and f) optionally repeating steps c), d), and e) with additional partially or completely unprotected peptide segments, wherein if said steps c) and d) are repeated, the peptide segment of step e) prior to such repetition comprises a thioacid at its C-terminus.
 14. A method of preparing an assembled peptide comprising: a) binding a partially or completely unprotected first peptide segment to a solid phase via a linker, wherein said first peptide segment comprises an N-terminal cysteine and a C-terminal residue capable of binding to said linker, wherein said linker comprises a cleavable moiety and said first peptide segment is bound to said linker at said C-terminal residue; b) ligating a partially or completely unprotected second peptide segment to said solid phase-bound first peptide segment, wherein said second peptide segment comprises a thioester at its C-terminus, and wherein said C-terminal thioester of said second peptide segments binds to said N-terminal cysteine of said solid phase-bound first peptide segment to form a solid phase-bound peptide; c) removing said protecting groups; d) optionally repeating steps b) and c) at least once with another peptide segment, wherein if said steps b) and c) are repeated, the peptide segment of step b) prior to such repetition comprises a cysteine at its N-terminus, said cysteine being capable of binding to a protective group; and e) cleaving said cleavable moiety to release said assembled peptide from said solid phase.
 15. The method of claim 14, further comprising: monitoring the ligation reactions of said ligating using mass spectrometric analysis of the solid phase-bound peptides.
 16. A method of preparing an assembled peptide comprising: a) binding a partially or completely unprotected first peptide segment to a solid phase via a linker, wherein said first peptide segment comprises an N-terminal cysteine and a C-terminal residue capable of binding to said linker, wherein said linker comprises a cleavable moiety and said first peptide segment is bound to said linker at said C-terminal residue; and b) ligating a second peptide segment to said first peptide segment bound to said solid phase in aqueous solution, wherein said second peptide segment comprises a C-terminal thioester, wherein said C-terminal thioester of said second peptide segment binds to said N-terminal cysteine of said solid phase-bound first peptide segment to form a solid phase-bound assembled peptide optionally comprising a protected cysteine at its N-terminus.
 17. A method of preparing an assembled peptide comprising: a) binding a partially or completely unprotected first peptide segment to a solid phase via a linker to form a solid phase-bound peptide, b) ligating at least one partially or completely unprotected second peptide segment to said solid phase-bound peptide in aqueous solution to form a solid phase-bound assembled peptide, and c) cleaving said linker to release said assembled peptide.
 18. The method of claim 17, wherein said solid phase is water-compatible.
 19. The method of claim 17, wherein said aqueous solution comprises 1-8 M urea.
 20. The method of claim 17, wherein said aqueous solution comprises 1-6 M guanidine.HCl.
 21. The method of claim 17, wherein said aqueous solution comprises 10-60% acetonitrile in water.
 22. The method of claim 17, wherein said aqueous solution comprises a mixed aqueous/organic solvent.
 23. The method of claim 1, wherein said method is repeated with a plurality of partially or completely unprotected first peptides, such that said method produces a polypeptide library comprised of solid phase sequentially ligated assembled peptides.
 24. A method of producing an assembled peptide in the N-terminal to C-terminal direction, said method comprising the steps; a) ligating a partially or completely unprotected peptide segment having an N-terminal cysteine and a C-terminal thioacid in aqueous solution to a solid phase-bound peptide segment having a C-terminal thioester, to form a solid phase-bound peptide segment having a C-terminal thioacid; and b) converting said C-terminal thioacid of said solid phase-bound peptide to a C-terminal thioester, to form a solid phase-bound assembled peptide having a C-terminal thioester.
 25. The method of claim 24, wherein said solid phase-bound peptide segment comprises a solid phase bound to said peptide segment via a linker.
 26. The method of claim 25, wherein said linker is a cleavable linker.
 27. The method of claim 20, which further comprises releasing said assembled peptide from said solid phase by cleaving said cleavable linker.
 28. The method of claim 24, which further comprises ligating said solid phase-bound assembled peptide having a C-terminal thioester to a peptide segment having an N-terminal cysteine and a C-terminal group other than a thioacid, to form a solid phase-bound assembled peptide having a C-terminal group other than a thioacid.
 29. The method of claim 28, wherein said solid phase-bound assembled peptide segment comprises a solid phase bound to said peptide segment via a linker.
 30. The method of claim 29, wherein said linker is a cleavable linker.
 31. The method of claim 30, which further comprises releasing said assembled peptide from said solid phase by cleaving said cleavable linker.
 32. A method of producing an assembled peptide in the C-terminal to N-terminal direction, said method comprising the steps: a) ligating a partially or completely unprotected first peptide segment to a partially or completely unprotected second peptide segment in aqueous solution, said first peptide segment having an unprotected N-terminal cysteine and a C-terminal group bonded to a solid phase via a linker adjoined to said C-terminal group, said second peptide segment having an unprotected C-terminal thioester and optionally a protected N-terminal cysteine; b) deprotecting said protected N-terminal cysteine of said second peptide segment, if present, and ligating a third peptide segment having an unprotected C-terminal thioester to said unprotected N-terminal cysteine of said second peptide segment; and c) recovering said produced assembled peptide.
 33. The method of claim 32, wherein said third peptide comprises a protected N-terminal cysteine, and wherein steps b) and c) are repeated one or more times with one or more additional peptide segments compatible with said deprotecting and said ligating to produce said assembled peptide.
 34. The method of claim 32, wherein said linker is a cleavable linker.
 35. The method of claim 34, which further comprises releasing said assembled peptide from said solid phase by cleaving said cleavable linker.
 36. The method of claim 33, wherein said linker is a cleavable linker.
 37. The method of claim 36, which further comprises releasing said assembled peptide from said solid phase by cleaving said cleavable linker.
 38. A method of preparing an assembled peptide comprising: a) binding a partially or completely unprotected first peptide segment to a solid phase via a linker to form a solid phase-bound peptide, b) ligating at least a second peptide segment to said solid phase-bound peptide in aqueous solution that comprises 1-8 M urea to form a solid phase-bound assembled peptide, and c) cleaving said linker to release said assembled peptide.
 39. A method of preparing an assembled peptide comprising: a) binding a partially or completely unprotected first peptide segment to a solid phase via a linker to form a solid phase-bound peptide, b) ligating at least a second peptide segment to said solid phase-bound peptide in aqueous solution that comprises 10-60% acetonitrile in water to form a solid phase-bound assembled peptide, and c) cleaving said linker to release said assembled peptide.
 40. A method of preparing an assembled peptide comprising: a) binding a partially or completely unprotected first peptide segment to a solid phase via a linker to form a solid phase-bound peptide, b) ligating at least a second peptide segment to said solid phase-bound peptide in aqueous solution that comprises 1-6 M guanidine.HCl to form a solid phase-bound assembled peptide, and c) cleaving said linker to release said assembled peptide.
 41. The method of claim 17, wherein said aqueous solution comprises a neutral pH of about 6.0 to 8.0.
 42. The method of claim 17, wherein said first peptide segment comprises one or more amino acids having an unprotected side-chain functional group.
 43. The method of claim 17, wherein said second peptide segment comprises one or more amino acids having an unprotected side-chain functional group.
 44. The method of claim 17, wherein said solid phase is capable of swelling in said aqueous solution.
 45. The method of claim 17, wherein said linker is stable to said ligating in said aqueous solution. 